Cell-reactive, long-acting, or targeted compstatin analogs and related compositions and methods

ABSTRACT

Long-acting compstatin analogs comprising a clearance-reducing moiety attached to two compstatin analog moieties, each compstatin analog moiety comprising a cyclic peptide extended by a lysine residue or a sequence comprising a lysine residue at the N-terminus, C-terminus, or both, wherein the lysine residue is separated from the cyclic portion of the peptide by a rigid or flexible spacer comprising an oligo(ethylene glycol) moiety, the clearance reducing moiety comprises a linear polymer, wherein each end of the linear polymer is linked to one of the compstatin analog moieties by way of a carbamate, wherein (i) the spacer comprises —(CH2) m — and —(O—CH2-CH2—) n  joined covalently, wherein m is between 1 and 10 and n is between 1 and 10; and/or (ii) the spacer comprises 8-amino-3,6-dioxaoctanoic acid (AEEAc) or 11-amino-3,6,9-trioxaundecanoic acid.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. provisional patentapplication No. 61/727,094, filed Nov. 15, 2012, the entire contents ofwhich are hereby incorporated by reference.

SEQUENCE LISTING

The present specification makes reference to a Sequence Listing(submitted electronically as a .txt file named“2008575-0086_Sequence_Listing” on Nov. 10, 2017. The .txt file wasgenerated on Nov. 10, 2017 and is 42 kilobytes in size. The entirecontents of the Sequence Listing are herein incorporated by reference.

BACKGROUND OF THE INVENTION

Complement is a system consisting of more than 30 plasma and cell-boundproteins that plays a significant role in both innate and adaptiveimmunity. The proteins of the complement system act in a series ofenzymatic cascades through a variety of protein interactions andcleavage events. Complement activation occurs via three main pathways:the antibody-dependent classical pathway, the alternative pathway, andthe mannose-binding lectin (MBL) pathway. Inappropriate or excessivecomplement activation is an underlying cause or contributing factor to anumber of serious diseases and conditions, and considerable effort hasbeen devoted over the past several decades to exploring variouscomplement inhibitors as therapeutic agents. However, there remains aneed for innovative approaches to inhibiting complement activation for avariety of therapeutic purposes.

SUMMARY OF THE INVENTION

In some aspects, the invention provides cell-reactive compstatinanalogs. For example, the invention provides cell-reactive compstatinanalogs, compositions comprising cell-reactive compstatin analogs, andmethods of making, identifying, characterizing, and/or usingcell-reactive compstatin analogs. In some aspects, the inventionprovides a physiologically acceptable composition comprising acell-reactive compstatin analog. In some aspects, the invention providesa pharmaceutical grade composition comprising a cell-reactive compstatinanalog.

In some aspects, the invention provides long-acting compstatin analogs.For example, the invention provides long-acting compstatin analogs,compositions comprising long-acting compstatin analogs, and methods ofmaking, identifying, characterizing, and/or using long-acting compstatinanalogs. In some aspects, the invention provides a physiologicallyacceptable composition comprising a long-acting compstatin analog. Insome aspects, the invention provides a pharmaceutical grade compositioncomprising a long-acting compstatin analog.

In some aspects, the invention provides targeted compstatin analogs. Forexample, the invention provides targeted compstatin analogs,compositions comprising targeted compstatin analogs, and methods ofmaking, identifying, characterizing, and/or using targeted compstatinanalogs. In some aspects, the invention provides a physiologicallyacceptable composition comprising a targeted compstatin analog. In someaspects, the invention provides a pharmaceutical grade compositioncomprising a targeted compstatin analog.

The invention further provides methods of protecting a cell fromcomplement-mediated damage. In some embodiments, the methods comprisecontacting the cell with a cell-reactive compstatin analog. The cell canbe any type of cell in various embodiments. For example, in someembodiments, the cell is a blood cell. In some embodiments the bloodcell is a red blood cell (RBC), also referred to as an erythrocyte. Insome embodiments, the cell has abnormally low expression, surfacedensity, and/or activity of one or more complement regulatory proteins.For example, the cell may have a mutation in a gene encoding suchprotein, wherein the mutation results in reduced or absent expressionand/or reduced activity of the encoded protein. The cell can be of anyanimal type or species in various embodiments. For example, the cell canbe mammalian, e.g., primate (human or a non-human primate), rodent(e.g., mouse, rat, rabbit), ungulate (e.g., pig, sheep, cow), canine, orfeline. In many embodiments, the protection is from primate complement,e.g., human complement. In some embodiments, the cell is contacted exvivo (outside the body of a subject). In some embodiments the cell iscontacted in vivo (in a subject, e.g., a human). In some embodiments thecell is to be transplanted into a subject or has been transplanted intoa subject. In some aspects, the invention provides an isolated cellhaving a compstatin analog covalently attached thereto. In some aspects,the invention provides an isolated tissue or organ having a compstatinanalog attached to at least some of its cells.

The invention provides methods of treating a subject in need oftreatment for a complement-mediated disorder. In some embodiments themethod comprises administering a cell-reactive compstatin analog to thesubject. In some embodiments the method comprises administering along-acting compstatin analog to the subject. In some embodiments, along-acting compstatin analog is a cell-reactive compstatin analog. Insome embodiments, the complement-mediated disorder is paroxysmalnocturnal hemoglobinuria (PNH), atypical hemoloytic uremic syndrome(aHUS), or another disorder associated with complement-mediatedhemolysis. In some embodiments the disorder is ischemia/reperfusion(I/R) injury (e.g., due to myocardial infarction, thromboembolic stroke,or surgery. In some embodiments, the disorder is trauma. In someembodiments, the disorder is transplant rejection. In some embodimentsthe disorder is a chronic respiratory disorder, e.g., asthma or COPD.

All articles, books, patent applications, patents, other publications,websites, and databases mentioned in this application are incorporatedherein by reference. In the event of a conflict between thespecification and any of the incorporated references the specification(including any amendments thereto) shall control. Unless otherwiseindicated, art-accepted meanings of terms and abbreviations are usedherein. The practice of certain aspects described herein may employconventional techniques of molecular biology, cell culture, recombinantnucleic acid (e.g., DNA) technology, immunology, and/or nucleic acid andpolypeptide synthesis, detection, manipulation, and quantification,etc., that are within the ordinary skill of the art. See, e.g., Ausubel,F., et al., (eds.), Current Protocols in Molecular Biology, CurrentProtocols in Immunology, Current Protocols in Protein Science, andCurrent Protocols in Cell Biology, all John Wiley & Sons, N.Y., e.g.,edition current as of January 2010 or later; Sambrook, Russell, andSambrook, Molecular Cloning: A Laboratory Manual, 3^(rd) ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, 2001 or 4^(th) ed,2012.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a plot that shows percent complement activation inhibitingactivity of compstatin analog CA28 (SEQ ID NO: 28) and three long-actingcompstatin analogs (CA28-1, CA28-2, CA28-3), as a function of peptideconcentration (μM). Inhibition of complement activation was tested invitro using a classical complement inhibition assay. The plot showsvalues obtained by averaging the results of two sets of measurements.CA28 (circles), CA28-1 (crosses (x)); CA28-2 (triangles), CA28-3(squares).

FIG. 2 is a plot that shows percent complement activation inhibitingactivity of CA28 and long-acting compstatin analogs CA28-2 and CA28-3,as a function of compound concentration (μM). CA28 (squares), CA28-2(diamonds), CA28-3 (circles). CA28-3 is a compound that containsmultiple peptide moieties. Although the activity per peptide moiety isless than the activity of an individual CA28 molecule, the totalactivity of CA28-3 exceeds the activity of CA28 on a molar basis.

FIG. 3 is a plot that shows plasma concentrations versus time of CA28and long-acting compstatin analogs CA28-2 and CA28-3 in Cynomolgusmonkeys following a single intravenous injection. CA28 was administeredat 200 mg/kg. CA28-2 and CA28-3 were each administered at 50 mg/kg. Incalculating the doses for these experiments the administered CA28-2 andCA28-3 substance was assumed to consist 80% of active compound w/w basedon dry weight. However, during the sample analysis, the standard curveassumed a 100% of active compound w/w based on dry weight, by anestimated 30%. Thus, the values for Cmax overestimate the actual Cmax.CA28 (squares), CA28-2 (triangles), CA28-3 (circles).

FIG. 4 is a plot that shows percent complement activation inhibitingactivity of CA28 and long-acting compstatin analogs CA28-4, as afunction of compound concentration (μM). Inhibition of complementactivation was tested in vitro using a classical complement inhibitionassay. The plot shows values obtained by averaging the results of foursets of measurements for CA28-4. CA28 (squares), CA28-4 (crosses).

FIG. 5 is a plot that shows concentrations versus time of CA28 andlong-acting compstatin analogs CA28-2, CA28-3, and CA28-4 in Cynomolgusmonkeys following a single intravenous injection. CA28 was administeredat 200 mg/kg. CA28-2, CA28-3, and CA28-4 were each administered at 50mg/kg. In calculating the doses for these experiments the administeredCA28-2 and CA28-3 substance was assumed to consist 80% of activecompound w/w based on dry weight. However, during the sample analysis,the standard curve assumed a 100% of active compound w/w based on dryweight. Thus, the values for Cmax overestimate the Cmax that would beachieved if these compounds had been administered at the indicated doseson a dry mass basis, by an estimated 30%. CA28 (squares), CA28-2(triangles), CA28-3 (circles), CA28-4 (inverted triangles).

FIG. 6 is a representative chromatogram showing ultraviolet (UV)detection of a PEG-based long-acting compstatin analog using reversephase HPLC. The peak with a retention time (RT) of 33.68 minutesrepresents the PEGylated compstatin analog and had a relative area of96%.

FIG. 7 is a plot that shows percent complement activation inhibitingactivity of CA28 and long-acting compstatin analogs, as a function ofcompound concentration (μM). CA28-2CS (diamonds); CA28-2GS (crosses);CA28-2HS (triangles); CA28-2TS (squares).

FIG. 8 is a plot that shows percent complement activation inhibitingactivity of CA28 and bifunctionalized long-acting compstatin analog,CA28-2GS-BF, as a function of compound concentration (micromoles). CA28(open circles); CA28-2G-SBF (filled circles)

FIG. 9 is a plot that shows the plasma concentration versus time of CA28and long-acting compstatin analog CA28-2GS-BF in Cynomolgus monkeysfollowing either a single intravenous injection (CA28 (squares) andCA28-2GS-BF (circles) or when administered by subcutaneous injectiononce daily for 7 days (CA28-2GS-BF only, asterisks). CA28-2GSBF wasadministered at 25 mg/ml. Dosing volume was 2 ml/kg for IV and 0.28ml/kg/day for the subcutaneous administration. Data for CA28 was from adifferent experiment in which the compound was also in 5% dextrose andwas formulated as 20 mg/ml with a 10 ml/kg dosing volume. The vehicle ineach case was 5% dextrose in water.

FIGS. 10(A) and 10(B) presents plots that show percent complementactivation inhibiting activity of CA28 and bifunctionalized long-actingcompstatin analog, CA28-2TS-BF, as a function of compound concentration(micromoles). (A) Classical pathway inhibition by CA28 (circles) andCA28-2TS-BF (crosses). (B) Alternative pathway inhibition. CA28(circles) and CA28-2TS-BF (crosses).

FIG. 10(C) (assuming a PEG moiety of 40 kD) shows the structure ofCA28-2TS-BF.

FIG. 11 is a plot that shows the plasma concentration versus time ofCA28 and long-acting compstatin analog CA28-2TS-BF in Cynomolgus monkeysfollowing a single intravenous injection of CA28 at 200 mg/kg (squares),a single intravenous injection of CA28-2TS-BF at 7 mg/kg (asterisks),subcutaneous injection of CA28-2TS-BF at 7 mg/kg once only (circles) orsubcutaneous injection of CA28-2TS-BF at 7 mg/kg once daily for 7consecutive days (inverted triangles). The vehicle in each case was 5%dextrose in water.

FIG. 12 shows flow cytometric analysis of C3 deposition on red bloodcells from a patient with PNH, which were exposed to activatedcomplement in a modified Ham's test. (A) Results of a dilutionexperiment demonstrating the effect of CA28 on C3 deposition are shown.(B) Results of a dilution experiment demonstrating the effect ofCA28-2GS-BF on C3 deposition are shown. Compound concentrations used areshown on and above each panel.

FIG. 13 shows flow cytometric analysis of C3 deposition on red bloodcells from a patient with PNH, which were exposed to activatedcomplement in a modified Ham's test in the absence of complementinhibitors (left panel), in the presence of anti-C5 monoclonal antibodyeculizumab (middle panel) and in the presence of CA28-2GS-BF (rightpanel).

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION I.Definitions

The terms “approximately” or “about” in reference to a number generallyinclude numbers that fall within ±10%, in some embodiments ±5%, in someembodiments ±1%, in some embodiments ±0.5% of the number unlessotherwise stated or otherwise evident from the context (except wheresuch number would impermissibly exceed 100% of a possible value).

A “complement component” or “complement protein” is a protein that isinvolved in activation of the complement system or participates in oneor more complement-mediated activities. Components of the classicalcomplement pathway include, e.g., C1q, C1r, C1s, C2, C3, C4, C5, C6, C7,C8, C9, and the C5b-9 complex, also referred to as the membrane attackcomplex (MAC) and active fragments or enzymatic cleavage products of anyof the foregoing (e.g., C3a, C3b, C4a, C4b, C5a, etc.). Components ofthe alternative pathway include, e.g., factors B, D, and properdin.Components of the lectin pathway include, e.g., MBL2, MASP-1, andMASP-2. Complement components also include cell-bound receptors forsoluble complement components, wherein such receptor mediates one ormore biological activities of such soluble complement componentfollowing binding of the soluble complement component. Such receptorsinclude, e.g., C5a receptor (C5aR), C3a receptor (C3aR), ComplementReceptor 1 (CR1), Complement Receptor 2 (CR2), Complement Receptor 3(CR3, also known as CD45), etc. It will be appreciated that the term“complement component” is not intended to include those molecules andmolecular structures that serve as “triggers” for complement activation,e.g., antigen-antibody complexes, foreign structures found on microbialor artificial surfaces, etc.

A “complement-mediated disorder” is any disorder in which complementactivation is known or suspected of being a contributing and/or at leastpartially causative factor in at least some subjects suffering from thedisorder, e.g., disorders in which complement activation results intissue damage. Non-limiting examples of complement-mediated disordersinclude, but are not limited to, (i) various disorders characterized byhemolysis or hemolytic anemia such as atypical hemolytic uremicsyndrome, cold agglutinin disease, paroxysmal nocturnal hemoglobinuria,transfusion reactions; (ii) transplant rejection (e.g., hyperacute oracute transplant rejection) or transplant dysfunction; (iii) disordersinvolving ischemia/reperfusion injury such as trauma, surgery (e.g.,aneurysm repair), myocardial infarction, ischemic stroke; (iv) disordersof the respiratory system such as asthma and chronic obstructivepulmonary disease (COPD); (v) arthritis, e.g., rheumatoid arthritis;(vi) ocular disorders such as age-related macular degeneration (AMD),diabetic retinopathy, glaucoma, and uveitis. “Disorder” is usedinterchangeably herein with “disease”, “condition”, and similar words torefer to any impairment of health or state of abnormal functioning of anorganism, e.g., any state in which medical and/or surgical management isindicated or for which a subject appropriately seeks medical and/orsurgical attention. It should also be understood that the listing of aparticular disorder within a particular category is for convenience andis not intended to limit the invention. It will be understood thatcertain disorders could appropriately be listed in multiple categories.

A “complement regulatory protein” is a protein involved in regulatingcomplement activity. A complement regulatory protein may down-regulatecomplement activity by, e.g., inhibiting complement activation or byinactivating or accelerating decay of one or more activated complementproteins. Examples of complement regulatory proteins include C1inhibitor, C4 binding protein, clusterin, vitronectin, CFH, factor I,and the cell-bound proteins CD46, CD55, CD59, CR1, CR2, and CR3.

“Isolated”, as used herein, means 1) separated from at least some of thecomponents with which it is usually associated in nature; 2) prepared orpurified by a process that involves the hand of man; and/or 3) notoccurring in nature, e.g., present in an artificial environment. Ingeneral, unless otherwise indicated or clearly evident, any entity,product, agent, composition, etc., may be deemed “isolated”, if desired.

“Linked”, as used herein with respect to two or more moieties, meansthat the moieties are physically associated or connected with oneanother to form a molecular structure that is sufficiently stable sothat the moieties remain associated under the conditions in which thelinkage is formed and, preferably, under the conditions in which the newmolecular structure is used, e.g., physiological conditions. In certainpreferred embodiments of the invention the linkage is a covalentlinkage. In other embodiments the linkage is noncovalent. Moieties maybe linked either directly or indirectly. When two moieties are directlylinked, they are either covalently bonded to one another or are insufficiently close proximity such that intermolecular forces between thetwo moieties maintain their association. When two moieties areindirectly linked, they are each linked either covalently ornoncovalently to a third moiety, which maintains the association betweenthe two moieties. In general, when two moieties are referred to as beinglinked by a “linking moiety” or “linking portion”, the linkage betweenthe two linked moieties is indirect, and typically each of the linkedmoieties is covalently bonded to the linking moiety. Two moieties may belinked using a “linker”. A linker can be any suitable moiety that reactswith the entities to be linked within a reasonable period of time, underconditions consistent with stability of the entities (portions of whichmay be protected as appropriate, depending upon the conditions), and insufficient amount, to produce a reasonable yield. Typically the linkerwill contain at least two functional groups, one of which reacts with afirst entity and the other of which reacts with a second entity. It willbe appreciated that after the linker has reacted with the entities to belinked, the term “linker” may refer to the part of the resultingstructure that originated from the linker, or at least the portion thatdoes not include the reacted functional groups. A linking moiety maycomprise a portion that does not participate in a bond with the entitiesbeing linked, and whose main purpose may be to spatially separate theentities from each other. Such portion may be referred to as a “spacer”.

As used herein, “physiological conditions” refers to a set of conditionssuch as temperature, salt concentration, pH that at least in part mimicthose conditions as typically found in a living subject, e.g., amammalian subject. In some aspects, physiological conditions refer toconditions in an aqueous medium, e.g., a medium comprising at least 90%,95%, 96%, 97%, 97%, 99%, or about 100% water on a volume/volume basis.In some embodiments other liquids, if present, do not substantiallyaffect protein secondary or tertiary structure. In some embodimentsphysiological conditions at least in part mimic those found in a bodyfluid such as blood or extracellular fluid, e.g., interstitial fluid,e.g., of a mammalian subject. A variety of physiological conditionsuseful for, e.g., in vitro assays, are known in the art. Generally, amedium under physiological conditions contains a physiologicalconcentration of salt, e.g., sodium chloride. In some embodiments aphysiological concentration of salt refers to a concentration rangingfrom about 250 mOsm/L to about 350 mOsm/L, e.g., about 275 mOsm/L toabout 325 mOsm/L, e.g., about 300 mOsm/L. In some embodimentsphysiological conditions are approximately isotonic to a body fluid,e.g., blood or extracellular fluid, e.g., interstitial fluid. In someembodiments physiological conditions include a pH ranging from about 6.5to about 7.8, e.g., about 7.0 to about 7.5. In some embodiments aphysiological medium comprises a buffer substance that helps maintainthe pH of the medium within a physiological range. In some embodimentsphysiological conditions comprise conditions such that a typicalmammalian protein, e.g., a protein typically found in a body fluid, suchas blood or extracellular fluid, substantially retains the secondaryand, if applicable, tertiary structure that such protein has in the bodyfluid in which it is normally found. In some embodiments components of aphysiological medium are typically substantially non-toxic to mammaliancells at the concentration at which they are present in thephysiological medium. A variety of physiological media (sometimes termed“buffers”) are listed in various standard references, such as thosecited above (e.g., Sambrook, et al., Protocols series). In someembodiments a physiological temperature ranges from about 25 degrees C.to about 38 degrees C., e.g., from about 30 degrees C. to about 37degrees C., e.g., 35 degrees C. to 37 degrees C.

“Polypeptide”, as used herein, refers to a polymer of amino acids,optionally including one or more amino acid analogs. A protein is amolecule composed of one or more polypeptides. A peptide is a relativelyshort polypeptide, typically between about 2 and 60 amino acids inlength, e.g., between 8 and 40 amino acids in length. The terms“protein”, “polypeptide”, and “peptide” may be used interchangeably.Polypeptides used herein may contain amino acids such as those that arenaturally found in proteins, amino acids that are not naturally found inproteins, and/or amino acid analogs that are not amino acids. As usedherein, an “analog” of an amino acid may be a different amino acid thatstructurally resembles the amino acid or a compound other than an aminoacid that structurally resembles the amino acid. A large number ofart-recognized analogs of the 20 amino acids commonly found in proteins(the “standard” amino acids) are known. One or more of the amino acidsin a polypeptide may be modified, for example, by the addition of achemical entity such as a carbohydrate group, a phosphate group, afarnesyl group, an isofarnesyl group, a fatty acid group, a linker forconjugation, functionalization, or other modification, etc. Certainnon-limiting suitable analogs and modifications are described inWO2004026328 and/or below. The polypeptide may be acetylated, e.g., atthe N-terminus and/or amidated, e.g., at the C-terminus.

The term “purified”, as used herein, refers to substances that have beenseparated from at least some or most of the components with which theyare associated in nature or when originally generated or with which theywere associated prior to purification. In general, such purificationinvolves action of the hand of man. Purified agents may be partiallypurified, substantially purified, or pure. Such agents may be, forexample, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or more than 99% pure. In some embodiments, a nucleic acid,polypeptide, or small molecule is purified such that it constitutes atleast 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, of the totalnucleic acid, polypeptide, or small molecule material, respectively,present in a preparation. In some embodiments, an organic substance,e.g., a nucleic acid, polypeptide, or small molecule, is purified suchthat it constitutes at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or more, of the total organic material present in a preparation.Purity may be based on, e.g., dry weight, size of peaks on achromatography tracing (GC, HPLC, etc.), molecular abundance,electrophoretic methods, intensity of bands on a gel, spectroscopic data(e.g., NMR), elemental analysis, high throughput sequencing, massspectrometry, or any art-accepted quantification method. In someembodiments, water, buffer substances, ions, and/or small molecules(e.g., synthetic precursors such as nucleotides or amino acids), canoptionally be present in a purified preparation. A purified agent may beprepared by separating it from other substances (e.g., other cellularmaterials), or by producing it in such a manner to achieve a desireddegree of purity. In some embodiments “partially purified” with respectto a molecule produced by a cell means that a molecule produced by acell is no longer present within the cell, e.g., the cell has been lysedand, optionally, at least some of the cellular material (e.g., cellwall, cell membrane(s), cell organelle(s)) has been removed and/or themolecule has been separated or segregated from at least some moleculesof the same type (protein, RNA, DNA, etc.) that were present in thelysate.

“Recombinant host cells”, “host cells”, and other such terms, denoteprokaryotic or eukaryotic cells or cell lines that contain an exogenousnucleic acid (typically DNA) such as an expression vector comprising anucleic acid that encodes a polypeptide of interest. It will beunderstood that such terms include the descendants of the originalcell(s) into which the vector or other nucleic acid has been introduced.Appropriate host cells include any of those routinely used in the artfor expressing polynucleotides (e.g., for purposes of producingpolypeptide(s) encoded by such polynucleotides) including, for example,prokaryotes, such as E. coli or other bacteria such as species ofEscherichia; Lactobacillus, Bacillus (e.g., B. subtilis), SalmonellaPseudomonas, Streptomyces, Staphylococcus, etc.; and eukaryotes,including for example, fungi, such as yeast (e.g., Pichia (e.g., Pichiapastoris), Kluyveromyces, such as K. lactis, Hansenula, e.g. H.polymorpha). Examples of other fungal cells are cells of filamentousfungi, e.g. Aspergillus spp., Neurospora spp., Fusarium spp. orTrichoderma spp., e.g., strains of A. oryzae, A. nidulans or A. niger;insect cells (e.g., Sf9), plant cells, and animal cells, e.g., mammaliancells such as CHO, R1.1, B-W, L-M, African Green Monkey Kidney cells(e.g. COS-1, COS-7, BSC-1, BSC-40 and BMT-10), and cultured human cells.Also encompassed are genetically modified cells in genetically modified(e.g., transgenic) plants or animals, wherein a recombinant polypeptideis produced by at least some such cells. A polypeptide may be secretedin milk, harvested from plant material, etc. The exogenous nucleic acidmay be stably maintained as an episome such as a plasmid or may at leastin part be integrated into the host cell's genome, optionally afterbeing copied or reverse transcribed. Terms such as “host cells”, etc.,are also used to refer to cells or cell lines that can be used asrecipients for an exogenous nucleic acid, prior to introduction of thenucleic acid. A “recombinant polynucleotide” generally is apolynucleotide that contains nucleic acid sequences that are not foundjoined directly to one another in nature. For example, the nucleic acidsequences may occur in different genes or different species or one ormore of the sequence(s) may be a variant of a naturally occurringsequence or may at least in part be an artificial sequence that is nothomologous to a naturally occurring sequence. A “recombinantpolypeptide” generally is a polypeptide that is at least in partproduced by transcription and translation of an exogenous nucleic acidby a recombinant host cell or by a cell-free in vitro expression systemand/or that contains amino acid sequences that are not found joineddirectly to one another in nature. In the latter case, the recombinantpolypeptide may be referred to as a “chimeric polypeptide”. The aminoacid sequences in a chimeric polypeptide may, for example, occur indifferent genes or in different species or one or more of thesequence(s) may be a variant of a naturally occurring sequence or may atleast in part be an artificial sequence that is not identical or in someembodiments is not homologous to a naturally occurring sequence over asubstantial portion of the length. It will be understood that a chimericpolypeptide may comprise two or more polypeptides. For example, firstand second polypeptides A and B of a chimeric polypeptide may bedirectly linked (A-B or B-A) or may be separated by a third polypeptideportion C (A-C-B or B-C-A). In some embodiments, portion C represents apolypeptide linker which may, for example, comprise multiple glycineand/or serine residues or any of a variety of other amino acids. In someembodiments, two or more polypeptides may be linked by non-polypeptidelinker(s). “Recombinant” as used herein encompasses in certainembodiments polypeptides produced by joining (e.g., chemicallyconjugating, enzymatically conjugating), shorter recombinantpolypeptides that may be produced in recombinant host cells. In someembodiments a recombinant polypeptide may comprise a signal sequencethat directs secretion of the polypeptide or a sequence that directs theexpressed polypeptide to a specific compartment or organelle. Suitablesequences are known in the art. Appropriate sequences for a host celltype of interest (e.g., bacterial, fungal, mammalian, plant, etc.) maybe selected. A signal sequence may be located at or near (e.g., withinup to 10-50 amino acids of) the N-terminus or C-terminus in someembodiments. In some embodiments a polypeptide comprises a tag. A tagmay be useful to facilitate detection and/or purification of a proteinthat contains it. Examples of tags include polyhistidine-tag (e.g.,6X-His tag) (SEQ ID NO: 69), glutathione-S-transferase, maltose bindingprotein, NUS tag, SNUT tag, Strep tag, epitope tags such as V5, HA, Myc,or FLAG. In some embodiments a protease cleavage site is located in theregion between the tag and the polypeptide, allowing the polypeptide tobe separated from the tag by exposure to the protease. In someembodiments a polynucleotide that encodes a recombinant polypeptide isat least in part codon optimized for expression in a host cell ofinterest (e.g., bacterial, fungal, mammalian, plant, etc.). A tag may belocated at or near (e.g., within up to 10-50 amino acids of) the N- orC-terminus of a polypeptide in various embodiments. A recombinantpolypeptide may be isolated, purified, etc., using any of a variety ofmethods. See, e.g., Sambrook, Protocols series, or other standardreferences. Methods of use may include, e.g., dialysis (e.g., usingmembranes having defined pore size), chromatography, precipitation, gelpurification, or affinity-based methods that may, in some embodiments,utilize a tag or a specific binding reagent such as an antibody.

“Reactive functional groups” as used herein refers to groups including,but not limited to, olefins, acetylenes, alcohols, phenols, ethers,oxides, halides, aldehydes, ketones, carboxylic acids, esters, amides,cyanates, isocyanates, thiocyanates, isothiocyanates, amines,hydrazines, hydrazones, hydrazides, diazo, diazonium, nitro, nitriles,mercaptans, sulfides, disulfides, sulfoxides, sulfones, sulfonic acids,sulfinic acids, acetals, ketals, anhydrides, sulfates, sulfenic acids,isonitriles, amidines, imides, imidates, nitrones, hydroxylamines,oximes, hydroxamic acids, thiohydroxamic acids, allenes, ortho esters,sulfites, enamines, ynamines, ureas, pseudoureas, semicarbazides,carbodiimides, carbamates, imines, azides, azo compounds, azoxycompounds, and nitroso compounds, N-hydroxysuccinimide esters,maleimides, sulfhydryls, and the like. Methods to prepare each of thesefunctional groups are well known in the art and their application to ormodification for a particular purpose is within the ability of one ofskill in the art (see, for example, Sandler and Karo, eds. ORGANICFUNCTIONAL GROUP PREPARATIONS, Academic Press, San Diego, 1989, andHermanson, G., Bioconjugate Techniques, 2nd ed., Academic Press, SanDiego, 2008).

“Specific binding” generally refers to a physical association between atarget polypeptide (or, more generally, a target molecule) and a bindingmolecule such as an antibody or ligand. The association is typicallydependent upon the presence of a particular structural feature of thetarget such as an antigenic determinant, epitope, binding pocket orcleft, recognized by the binding molecule. For example, if an antibodyis specific for epitope A, the presence of a polypeptide containingepitope A or the presence of free unlabeled A in a reaction containingboth free labeled A and the binding molecule that binds thereto, willreduce the amount of labeled A that binds to the binding molecule. It isto be understood that specificity need not be absolute but generallyrefers to the context in which the binding occurs. For example, it iswell known in the art that numerous antibodies cross-react with otherepitopes in addition to those present in the target molecule. Suchcross-reactivity may be acceptable depending upon the application forwhich the antibody is to be used. One of ordinary skill in the art willbe able to select antibodies or ligands having a sufficient degree ofspecificity to perform appropriately in any given application (e.g., fordetection of a target molecule, for therapeutic purposes, etc.). It isalso to be understood that specificity may be evaluated in the contextof additional factors such as the affinity of the binding molecule forthe target versus the affinity of the binding molecule for othertargets, e.g., competitors. If a binding molecule exhibits a highaffinity for a target molecule that it is desired to detect and lowaffinity for nontarget molecules, the antibody will likely be anacceptable reagent. Once the specificity of a binding molecule isestablished in one or more contexts, it may be employed in other,preferably similar, contexts without necessarily re-evaluating itsspecificity. In some embodiments, the affinity (as measured by theequilibrium dissociation constant, Kd) of two molecules that exhibitspecific binding is 10⁻³M or less, e.g., 10⁻⁴M or less, e.g., 10⁻⁵ M orless, e.g., 10⁻⁶M or less, 10⁻⁷M or less, 10⁻⁸M or less, or 10⁻⁹M orless under the conditions tested, e.g., under physiological conditions.

A “subject” treated according to the instant invention is typically ahuman, a non-human primate, or a lower animal (e.g., a mouse or rat),which expresses or contains at least some primate (e.g., human)complement component C3 and, optionally, one or more additional primatecomplement component(s). In some embodiments the subject is male. Insome embodiments the subject is female. In some embodiments the subjectis an adult, e.g., a human at least 18 years of age, e.g., between 18and 100 years of age. In some embodiments, a human subject is at least12 years of age. In some embodiments a subject is an adult, e.g., ahuman at least 18 years of age, e.g., between 18 and 100 years of age.In some embodiments a subject is at least 40, 45, 50, 55, 60, 65, 70,75, or 80 years of age. In some embodiments the subject is a child,e.g., a human between 0 and 4 years of age, or between 5 and 11 years ofage.

“Treating”, as used herein in regard to treating a subject, refers toproviding treatment, i.e., providing any type of medical or surgicalmanagement of a subject. The treatment can be provided in order toreverse, alleviate, inhibit the progression of, prevent or reduce thelikelihood of a disease, or in order to reverse, alleviate, inhibit orprevent the progression of, prevent or reduce the likelihood of one ormore symptoms or manifestations of a disease. “Prevent” refers tocausing a disease or symptom or manifestation of a disease not to occurfor at least a period of time in at least some individuals. Treating caninclude administering a compound or composition to the subject followingthe development of one or more symptoms or manifestations indicative ofa disease, e.g., in order to reverse, alleviate, reduce the severity of,and/or inhibit or prevent the progression of the disease and/or toreverse, alleviate, reduce the severity of, and/or inhibit or one ormore symptoms or manifestations of the disease. A compound orcomposition can be administered to a subject who has developed adisease, or is at increased risk of developing the disease relative to amember of the general population. A compound or composition can beadministered to a subject who has developed a disease and is atincreased risk of developing one or more particular symptoms ormanifestations of the disease or an exacerbation of the disease relativeto other individuals diagnosed with the disease, or relative to thesubject's typical or average risk for such symptom or manifestation orexacerbation. For example, the subject may have been exposed to a“trigger” that places the subject at increased risk (e.g., temporarilyincreased risk) of experiencing an exacerbation. A compound orcomposition can be administered prophylactically, i.e., beforedevelopment of any symptom or manifestation of the disease. Typically inthis case the subject will be at risk of developing the disease, e.g.,relative to a member of the general population, optionally matched interms of age, sex, and/or other demographic variable(s).

A “vector” may be any of a variety of nucleic acid molecules, viruses,or portions thereof that are capable of mediating entry of, e.g.,transferring, transporting, etc., a nucleic acid of interest betweendifferent genetic environments or into a cell. The nucleic acid ofinterest may be linked to, e.g., inserted into, the vector using, e.g.,restriction and ligation. Vectors include, for example, DNA or RNAplasmids, cosmids, naturally occurring or modified viral genomes orportions thereof, nucleic acids that can be packaged into viral capsids,mini-chromosomes, artificial chromosomes, etc. Plasmid vectors typicallyinclude an origin of replication (e.g., for replication in prokaryoticcells). A plasmid may include part or all of a viral genome (e.g., aviral promoter, enhancer, processing or packaging signals, and/orsequences sufficient to give rise to a nucleic acid that can beintegrated into the host cell genome and/or to give rise to infectiousvirus). Viruses or portions thereof that can be used to introducenucleic acids into cells may be referred to as viral vectors. Viralvectors include, e.g., adenoviruses, adeno-associated viruses,retroviruses (e.g., lentiviruses, vaccinia virus and other poxviruses,herpesviruses (e.g., herpes simplex virus), and others. Baculovirus areof use, e.g., in insect cells. A wide range of plant viral vectors areknown and include, e.g., those based on or comprising Cauliflower MosaicVirus, Tobacco Mosaic Virus, or one or more genetic elements thereof(e.g., Cauliflower Mosaic Virus 35S promoter). Viral vectors may or maynot contain sufficient viral genetic information for production ofinfectious virus when introduced into host cells, i.e., viral vectorsmay be replication-competent or replication-defective. In someembodiments, e.g., where sufficient information for production ofinfectious virus is lacking, it may be supplied by a host cell or byanother vector introduced into the cell, e.g., if production of virus isdesired. In some embodiments such information is not supplied, e.g., ifproduction of virus is not desired. A nucleic acid to be transferred maybe incorporated into a naturally occurring or modified viral genome or aportion thereof or may be present within a viral capsid as a separatenucleic acid molecule. A vector may contain one or more nucleic acidsencoding a marker suitable for identifying and/or selecting cells thathave taken up the vector. Markers include, for example, various proteinsthat increase or decrease either resistance or sensitivity toantibiotics or other agents (e.g., a protein that confers resistance toan antibiotic such as puromycin, hygromycin or blasticidin), enzymeswhose activities are detectable by assays known in the art (e.g.,β-galactosidase or alkaline phosphatase), and proteins or RNAs thatdetectably affect the phenotype of cells that express them (e.g.,fluorescent proteins). Vectors often include one or more appropriatelypositioned sites for restriction enzymes, which may be used tofacilitate insertion into the vector of a nucleic acid, e.g., a nucleicacid to be expressed. An expression vector is a vector into which adesired nucleic acid has been inserted or may be inserted such that itis operably linked to regulatory elements (also termed “regulatorysequences”, “expression control elements”, or “expression controlsequences”) and may be expressed as an RNA transcript (e.g., an mRNAthat can be translated into protein or a noncoding RNA). Expressionvectors include regulatory sequence(s), e.g., expression controlsequences, sufficient to direct transcription of an operably linkednucleic acid under at least some conditions; other elements required orhelpful for expression may be supplied by, e.g., the host cell or by anin vitro expression system. Such regulatory sequences typically includea promoter and may include enhancer sequences or upstream activatorsequences. In some embodiments a vector may include sequences thatencode a 5′ untranslated region and/or a 3′ untranslated region, whichmay comprise a cleavage and/or polyadenylation signal. In general,regulatory elements may be contained in a vector prior to insertion of anucleic acid whose expression is desired or may be contained in aninserted nucleic acid or may be inserted into a vector followinginsertion of a nucleic acid whose expression is desired. As used herein,a nucleic acid and regulatory element(s) are said to be “operablylinked” when they are covalently linked so as to place the expression ortranscription of the nucleic acid under the influence or control of theregulatory element(s). For example, a promoter region would be operablylinked to a nucleic acid if the promoter region were capable ofeffecting transcription of that nucleic acid. One of ordinary skill inthe art will be aware that the precise nature of the regulatorysequences useful for gene expression may vary between species or celltypes, but may in general include, as appropriate, sequences involvedwith the initiation of transcription, RNA processing, or initiation oftranslation. The choice and design of an appropriate vector andregulatory element(s) is within the ability and discretion of one ofordinary skill in the art. For example, one of skill in the art willselect an appropriate promoter (or other expression control sequences)for expression in a desired species (e.g., a prokaryotic (bacterial) oreukaryotic (e.g., fungal, plant, mammalian species) or cell type. Avector may contain a promoter capable of directing expression inmammalian cells, such as a suitable viral promoter, e.g., from acytomegalovirus (CMV), retrovirus, simian virus (e.g., SV40), papillomavirus, herpes virus or other virus that infects mammalian cells, or amammalian promoter from, e.g., a gene such as EF1alpha, ubiquitin (e.g.,ubiquitin B or C), globin, actin, phosphoglycerate kinase (PGK), etc.,or a composite promoter such as a CAG promoter (combination of the CMVearly enhancer element and chicken beta-actin promoter). In someembodiments a human promoter may be used. In some embodiments, apromoter that ordinarily directs transcription by a eukaryotic RNApolymerase I (a “pol I promoter”), e.g., (a U6, H1, 7SK or tRNA promoteror a functional variant thereof) may be used. In some embodiments, apromoter that ordinarily directs transcription by a eukaryotic RNApolymerase II (a “pol II promoter”) or a functional variant thereof isused. In some embodiments, a promoter that ordinarily directstranscription by a eukaryotic RNA polymerase III (a “pol III promoter”),e.g., a promoter for transcription of ribosomal RNA (other than 5S rRNA)or a functional variant thereof is used. One of ordinary skill in theart will select an appropriate promoter for directing transcription of asequence of interest. Examples of expression vectors that may be used inmammalian cells include, e.g., the pcDNA vector series, pSV2 vectorseries, pCMV vector series, pRSV vector series, pEF1 vector series,Gateway® vectors, etc. In some embodiments, regulatable (e.g., inducibleor repressible) expression control element(s), e.g., a regulatablepromoter, is/are used so that expression can be regulated, e.g., turnedon or increased or turned off or decreased. In some embodiments a vectormay comprise a polynucleotide sequence that encodes a polypeptide,wherein the polynucleotide sequence is positioned in frame with anucleic acid inserted into the vector so that an N- or C-terminal fusionis created. In some embodiments a polypeptide encoded by thepolynucleotide sequence may comprise a signal sequence (which directssecretion of a protein) or a sequence that directs the expressed proteinto a specific organelle or location in the cell such as the nucleus ormitochondria. In some embodiments a polypeptide comprises a tag. A tagmay be useful to facilitate detection and/or purification of a proteinthat contains it. Examples of tags include polyhistidine-tag (e.g.,6X-His tag) (SEQ ID NO: 69), glutathione-S-transferase, maltose bindingprotein, NUS tag, SNUT tag, Strep tag, epitope tags such as V5, HA, Myc,or FLAG. In some embodiments a protease cleavage site is located in theregion between the protein encoded by the inserted nucleic acid and thepolypeptide, allowing the polypeptide to be removed by exposure to theprotease. Vectors may be introduced into host cells using methods knownin the art. One of ordinary skill will select an appropriate methodbased, e.g., on the vector, cell type, etc. Examples of suitable methodsinclude, e.g., calcium phosphate-mediated transfection, transfectionwith any of a variety of commercially available reagents; e.g.,lipid-based or non-lipid based, such as FuGENE, Lipofectamine,TurboFect; electroporation; microparticle bombardment, etc. Such methodsare explained in detail in standard references such as Sambrook,Protocols series, and others.

As used herein the term “aliphatic” denotes a hydrocarbon moiety thatmay be straight-chain (i.e., unbranched), branched, or cyclic (includingfused, bridging, and spiro-fused polycyclic) and may be completelysaturated or may contain one or more units of unsaturation, but which isnot aromatic. Unless otherwise specified, aliphatic groups contain 1-30carbon atoms. In some embodiments, aliphatic groups contain 1-10 carbonatoms. In other embodiments, aliphatic groups contain 1-8 carbon atoms.In still other embodiments, aliphatic groups contain 1-6 carbon atoms,and in yet other embodiments aliphatic groups contain 1-4 carbon atoms.Suitable aliphatic groups include, but are not limited to, linear orbranched, alkyl, alkenyl, and alkynyl groups, and hybrids thereof suchas (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

As used herein, “alkyl” refers to a saturated straight, branched, orcyclic hydrocarbon having from about 1 to about 22 carbon atoms (and allcombinations and subcombinations of ranges and specific numbers ofcarbon atoms therein), with from about 1 to about 12, or about 1 toabout 7 carbon atoms being preferred in certain embodiments of theinvention. Alkyl groups include, but are not limited to, methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, cyclopentyl,isopentyl, neopentyl, n-hexyl, isohexyl, cyclohexyl, cyclooctyl,adamantyl, 3-methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl.

As used herein, “halo” refers to F, C1, Br or I.

As used herein, “alkanoyl” refers to an optionally substituted straightor branched aliphatic acyclic residue having about 1 to 10 carbon atoms(and all combinations and subcombinations of ranges and specific numberof carbon atoms) therein, e.g., from about 1 to 7 carbon atoms which, aswill be appreciated, is attached to a terminal C═O group with a singlebond (and may also be referred to as an “acyl group”). Alkanoyl groupsinclude, but are not limited to, formyl, acetyl, propionyl, butyryl,isobutyryl, pentanoyl, isopentanoyl, 2-methyl-butyryl,2,2-dimethoxypropionyl, hexanoyl, heptanoyl, octanoyl, and the like, andfor purposes of the present invention a formyl group is considered analkanoyl group. “Lower alkanoyl” refers to an optionally substitutedstraight or branched aliphatic acyclic residue having about 1 to about 5carbon atoms (and all combinations and subcombinations of ranges andspecific number of carbon atoms). Such groups include, but are notlimited to, formyl, acetyl, propionyl, butyryl, isobutyryl, pentanoyl,isopentanoyl, etc.

As used herein, “aryl” refers to an optionally substituted, mono- orbicyclic aromatic ring system having from about 5 to about 14 carbonatoms (and all combinations and subcombinations of ranges and specificnumbers of carbon atoms therein), with from about 6 to about 10 carbonsbeing preferred. Non-limiting examples include, for example, phenyl andnaphthyl.

As used herein, “aralkyl” refers to alkyl radicals bearing an arylsubstituent and having from about 6 to about 22 carbon atoms (and allcombinations and subcombinations of ranges and specific numbers ofcarbon atoms therein), with from about 6 to about 12 carbon atoms beingpreferred in certain embodiments. Aralkyl groups can be optionallysubstituted. Non-limiting examples include, for example, benzyl,naphthylmethyl, diphenylmethyl, triphenylmethyl, phenylethyl, anddiphenylethyl.

As used herein, the terms “alkoxy” and “alkoxyl” refer to an optionallysubstituted alkyl-O— group wherein alkyl is as previously defined.Exemplary alkoxy and alkoxyl groups include methoxy, ethoxy, n-propoxy,i-propoxy, n-butoxy, and heptoxy.

As used herein, “carboxy” refers to a —C(═O)OH group.

As used herein, “alkoxycarbonyl” refers to a —C(═O)O-alkyl group, wherealkyl is as previously defined.

As used herein, “aroyl” refers to a —C(═O)-aryl group, wherein aryl isas previously defined. Exemplary aroyl groups include benzoyl andnaphthoyl.

The term “cyclic ring system” refers to an aromatic or non-aromatic,partially unsaturated or fully saturated, 3- to 10-membered ring system,which includes single rings of 3 to 8 atoms in size and bi- andtri-cyclic ring systems which may include aromatic 5- or 6-membered arylor aromatic heterocyclic groups fused to a non-aromatic ring. Theseheterocyclic rings include those having from 1 to 3 heteroatomsindependently selected from the group consisting of oxygen, sulfur, andnitrogen. In certain embodiments, the term heterocyclic refers to anon-aromatic 5-, 6-, or 7-membered ring or a polycyclic group wherein atleast one ring atom is a heteroatom selected from the group consistingof 0, S, and N, including, but not limited to, a bi- or tri-cyclicgroup, comprising fused six-membered rings having between one and threeheteroatoms independently selected from the group consisting of theoxygen, sulfur, and nitrogen. In some embodiments, “cyclic ring system”refers to a cycloalkyl group which, as used herein, refers to groupshaving 3 to 10, e.g., 4 to 7 carbon atoms. Cycloalkyls include, but arenot limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl and the like, which, is optionally substituted. In someembodiments, “cyclic ring system” refers to a cycloalkenyl orcycloalkynyl moiety, which is optionally substituted.

Typically, substituted chemical moieties include one or moresubstituents that replace hydrogen. Exemplary substituents include, forexample, halo, alkyl, cycloalkyl, aralkyl, aryl, sulfhydryl, hydroxyl(—OH), alkoxyl, cyano (—CN), carboxyl (—COOH), —C(═O)O-alkyl,aminocarbonyl (—C(═O)NH₂), —N-substituted aminocarbonyl (—C(═O)NHR″),CF₃, CF₂CF₃, and the like. In relation to the aforementionedsubstituents, each moiety R″ can be, independently, any of H, alkyl,cycloalkyl, aryl, or aralkyl, for example.

As used herein, “L-amino acid” refers to any of the naturally occurringlevorotatory alpha-amino acids normally present in proteins or the alkylesters of those alpha-amino acids. The term “D-amino acid” refers todextrorotatory alpha-amino acids. Unless specified otherwise, all aminoacids referred to herein are L-amino acids.

As used herein, an “aromatic amino acid” is an amino acid that comprisesat least one aromatic ring, e.g., it comprises an aryl group.

As used herein, an “aromatic amino acid analog” is an amino acid analogthat comprises at least one aromatic ring, e.g., it comprises an arylgroup.

II. Overview

The present invention provides cell-reactive compstatin analogs andmethods of relating thereto, e.g., methods of use thereof. Cell-reactivecompstatin analogs are compounds that comprise a compstatin analogmoiety and a cell-reactive functional group that is capable of reactingwith a functional group exposed at the surface of a cell, e.g., underphysiological conditions, to form a covalent bond. The cell-reactivecompstatin analog thus becomes covalently attached to the cell. Withoutwishing to be bound by any particular theory, a cell-tethered compstatinanalog protects the cell from complement-mediated damage by, forexample, binding to C3 (which may be in the form of C3(H₂O)) at the cellsurface and/or in the vicinity of the cell and inhibiting C3 cleavageand activation, and/or by binding to C3b and inhibiting its depositionon the cell or participation in the complement activation cascade. Insome aspects of the invention, isolated cells are contacted with acell-reactive compstatin analog ex vivo (outside the body). In someaspects of the invention, the cells are present in an isolated tissue ororgan, e.g., a tissue or organ to be transplanted into a subject. Insome aspects of the invention, cells are contacted with a cell-reactivecompstatin analog in vivo, by administering the cell-reactive compstatinanalog to a subject. The cell-reactive compstatin analog becomescovalently attached to cells in vivo. In some aspects, the inventiveapproach protects cells, tissues, and/or organs from the deleteriouseffects of complement activation for at least two weeks, without needfor retreatment during that time.

In some aspects, the invention provides compstatin analogs comprising atargeting moiety that binds non-covalently to a target molecule presentat the surface of cells or tissues or to an extracellular substance notattached to cells or tissues. Such compstatin analogs are referred toherein as “targeted compstatin analogs”). Often the target molecule is aprotein or carbohydrate attached to the cell membrane and exposed at thecell surface. The targeting moiety targets the compstatin analog to acell, tissue, or location susceptible to complement activation. In someaspects of the invention, isolated cells are contacted with a targetedcompstatin analog ex vivo (outside the body). In some aspects of theinvention, the cells are present in an isolated tissue or organ, e.g., atissue or organ to be transplanted into a subject. In some aspects ofthe invention, a targeted compstatin analog is administered to a subjectand becomes non-covalently attached to a cell, tissue, or extracellularsubstance in vivo. In some aspects, the inventive approach protectscells, tissues, and/or organs from the deleterious effects of complementactivation for at least two weeks, without need for retreatment duringthat time. In some embodiments, a targeted compstatin analog comprisesboth a targeting moiety and a cell-reactive moiety. The targeting moietytargets the compstatin analog, e.g., to a particular cell type, bybinding non-covalently to a molecule on such cells. The cell-reactivemoiety then binds covalently to the cell or extracellular substance. Inother embodiments, a targeted compstatin analog does not comprise acell-reactive moiety.

In some aspects, the invention provides long-acting compstatin analogs,wherein the long-acting compstatin analogs comprise a moiety such aspolyethylene glycol (PEG) that prolongs the lifetime of the compound inthe body (e.g., by reducing its clearance from the blood). In someembodiments, a long-acting compstatin analog does not comprise atargeting moiety or a cell-reactive moiety. In some embodiments, along-acting compstatin analog comprises a targeting moiety and/or acell-reactive moiety.

III. Complement System

In order to facilitate understanding of the invention, and withoutintending to limit the invention in any way, this section provides anoverview of complement and its pathways of activation. Further detailsare found, e.g., in Kuby Immunology, 6^(th) ed., 2006; Paul, W. E.,Fundamental Immunology, Lippincott Williams & Wilkins; 6^(th) ed., 2008;and Walport M J., Complement. First of two parts. N Engl J Med.,344(14):1058-66, 2001.

Complement is an arm of the innate immune system that plays an importantrole in defending the body against infectious agents. The complementsystem comprises more than 30 serum and cellular proteins that areinvolved in three major pathways, known as the classical, alternative,and lectin pathways. The classical pathway is usually triggered bybinding of a complex of antigen and IgM or IgG antibody to C1 (thoughcertain other activators can also initiate the pathway). Activated C1cleaves C4 and C2 to produce C4a and C4b, in addition to C2a and C2b.C4b and C2a combine to form C3 convertase, which cleaves C3 to form C3aand C3b. Binding of C3b to C3 convertase produces C5 convertase, whichcleaves C5 into C5a and C5b. C3a, C4a, and C5a are anaphylotoxins andmediate multiple reactions in the acute inflammatory response. C3a andC5a are also chemotactic factors that attract immune system cells suchas neutrophils.

The alternative pathway is initiated by and amplified at, e.g.,microbial surfaces and various complex polysaccharides. In this pathway,hydrolysis of C3 to C3(H₂O), which occurs spontaneously at a low level,leads to binding of factor B, which is cleaved by factor D, generating afluid phase C3 convertase that activates complement by cleaving C3 intoC3a and C3b. C3b binds to targets such as cell surfaces and forms acomplex with factor B, which is later cleaved by factor D, resulting ina C3 convertase. Surface-bound C3 convertases cleave and activateadditional C3 molecules, resulting in rapid C3b deposition in closeproximity to the site of activation and leading to formation ofadditional C3 convertase, which in turn generates additional C3b. Thisprocess results in a cycle of C3 cleavage and C3 convertase formationthat significantly amplifies the response. Cleavage of C3 and binding ofanother molecule of C3b to the C3 convertase gives rise to a C5convertase. C3 and C5 convertases of this pathway are regulated by hostcell molecules CR1, DAF, MCP, CD59, and fH. The mode of action of theseproteins involves either decay accelerating activity (i.e., ability todissociate convertases), ability to serve as cofactors in thedegradation of C3b or C4b by factor I, or both. Normally the presence ofcomplement regulatory proteins on host cell surfaces preventssignificant complement activation from occurring thereon.

The C5 convertases produced in both pathways cleave C5 to produce C5aand C5b. C5b then binds to C6, C7, and C8 to form C5b-8, which catalyzespolymerization of C9 to form the C5b-9 membrane attack complex (MAC).The MAC inserts itself into target cell membranes and causes cell lysis.Small amounts of MAC on the membrane of cells may have a variety ofconsequences other than cell death.

The lectin complement pathway is initiated by binding of mannose-bindinglectin (MBL) and MBL-associated serine protease (MASP) to carbohydrates.The MB1-1 gene (known as LMAN-1 in humans) encodes a type I integralmembrane protein localized in the intermediate region between theendoplasmic reticulum and the Golgi. The MBL-2 gene encodes the solublemannose-binding protein found in serum. In the human lectin pathway,MASP-1 and MASP-2 are involved in the proteolysis of C4 and C2, leadingto a C3 convertase described above.

Complement activity is regulated by various mammalian proteins referredto as complement control proteins (CCPs) or regulators of complementactivation (RCA) proteins (U.S. Pat. No. 6,897,290). These proteinsdiffer with respect to ligand specificity and mechanism(s) of complementinhibition. They may accelerate the normal decay of convertases and/orfunction as cofactors for factor I, to enzymatically cleave C3b and/orC4b into smaller fragments. CCPs are characterized by the presence ofmultiple (typically 4-56) homologous motifs known as short consensusrepeats (SCR), complement control protein (CCP) modules, or SUSHIdomains, about 50-70 amino acids in length that contain a conservedmotif including four disulfide-bonded cysteines (two disulfide bonds),proline, tryptophan, and many hydrophobic residues. The CCP familyincludes complement receptor type 1 (CR1; C3b:C4b receptor), complementreceptor type 2 (CR2), membrane cofactor protein (MCP; CD46),decay-accelerating factor (DAF), complement factor H (fH), andC4b-binding protein (C4 bp). CD59 is a membrane-bound complementregulatory protein unrelated structurally to the CCPs. Complementregulatory proteins normally serve to limit complement activation thatmight otherwise occur on cells and tissues of the mammalian, e.g., humanhost. Thus, “self” cells are normally protected from the deleteriouseffects that would otherwise ensue were complement activation to proceedon these cells. Deficiencies or defects in complement regulatoryprotein(s) are involved in the pathogenesis of a variety ofcomplement-mediated disorders, e.g., as discussed herein.

IV. Compstatin Analogs

Compstatin is a cyclic peptide that binds to C3 and inhibits complementactivation. U.S. Pat. No. 6,319,897 describes a peptide having thesequence

(SEQ ID NO: 1) Ile-[Cys-Val-Val-Gln-Asp-Trp-Gly-His-His-Arg- Cys]-Thr,with the disulfide bond between the two cysteines denoted by brackets.It will be understood that the name “compstatin” was not used in U.S.Pat. No. 6,319,897 but was subsequently adopted in the scientific andpatent literature (see, e.g., Morikis, et al., Protein Sci.,7(3):619-27, 1998) to refer to a peptide having the same sequence as SEQID NO: 2 disclosed in U.S. Pat. No. 6,319,897, but amidated at the Cterminus as shown in Table 1 (SEQ ID NO: 8). The term “compstatin” isused herein consistently with such usage (i.e., to refer to SEQ ID NO:8). Compstatin analogs that have higher complement inhibiting activitythan compstatin have been developed. See, e.g., WO2004/026328(PCT/US2003/029653), Morikis, D., et al., Biochem Soc Trans. 32(Pt1):28-32, 2004, Mallik, B., et al., J. Med. Chem., 274-286, 2005;Katragadda, M., et al. J. Med. Chem., 49: 4616-4622, 2006; WO2007062249(PCT/US2006/045539); WO2007044668 (PCT/US2006/039397), WO/2009/046198(PCT/US2008/078593); WO/2010/127336 (PCT/US2010/033345) and discussionbelow.

Compstatin analogs may be acetylated or amidated, e.g., at theN-terminus and/or C-terminus. For example, compstatin analogs may beacetylated at the N-terminus and amidated at the C-terminus. Consistentwith usage in the art, “compstatin” as used herein, and the activitiesof compstatin analogs described herein relative to that of compstatin,refer to compstatin amidated at the C-terminus (Mallik, 2005, supra).

Concatamers or multimers of compstatin or a complement inhibiting analogthereof are also of use in the present invention.

As used herein, the term “compstatin analog” includes compstatin and anycomplement inhibiting analog thereof. The term “compstatin analog”encompasses compstatin and other compounds designed or identified basedon compstatin and whose complement inhibiting activity is at least 50%as great as that of compstatin as measured, e.g., using any complementactivation assay accepted in the art or substantially similar orequivalent assays. Certain suitable assays are described in U.S. Pat.No. 6,319,897, WO2004/026328, Morikis, supra, Mallik, supra, Katragadda2006, supra, WO2007062249 (PCT/US2006/045539); WO2007044668(PCT/US2006/039397), WO/2009/046198 (PCT/US2008/078593); and/orWO/2010/127336 (PCT/US2010/033345). The assay may, for example, measurealternative or classical pathway-mediated erythrocyte lysis or be anELISA assay. In some embodiments, an assay described in WO/2010/135717(PCT/US2010/035871) is used.

The activity of a compstatin analog may be expressed in terms of itsIC₅₀ (the concentration of the compound that inhibits complementactivation by 50%), with a lower IC₅₀ indicating a higher activity asrecognized in the art. The activity of a preferred compstatin analog foruse in the present invention is at least as great as that of compstatin.It is noted that certain modifications known to reduce or eliminatecomplement inhibiting activity and may be explicitly excluded from anyembodiment of the invention. The IC₅₀ of compstatin has been measured as12 μM using an alternative pathway-mediated erythrocyte lysis assay(WO2004/026328). It will be appreciated that the precise IC₅₀ valuemeasured for a given compstatin analog will vary with experimentalconditions (e.g., the serum concentration used in the assay).Comparative values, e.g., obtained from experiments in which IC₅₀ isdetermined for multiple different compounds under substantiallyidentical conditions, are of use. In one embodiment, the IC₅₀ of thecompstatin analog is no more than the IC₅₀ of compstatin. In certainembodiments of the invention the activity of the compstatin analog isbetween 2 and 99 times that of compstatin (i.e., the analog has an IC₅₀that is less than the IC₅₀ of compstatin by a factor of between 2 and99). For example, the activity may be between 10 and 50 times as greatas that of compstatin, or between 50 and 99 times as great as that ofcompstatin. In certain embodiments of the invention the activity of thecompstatin analog is between 99 and 264 times that of compstatin. Forexample, the activity may be 100, 110, 120, 130, 140, 150, 160, 170,180, 190, 200, 210, 220, 230, 240, 250, 260, or 264 times as great asthat of compstatin. In certain embodiments the activity is between 250and 300, 300 and 350, 350 and 400, or 400 and 500 times as great as thatof compstatin. The invention further contemplates compstatin analogshaving activities between 500 and 1000 times that of compstatin, ormore. In certain embodiments the IC₅₀ of the compstatin analog isbetween about 0.2 μM and about 0.5 μM. In certain embodiments the IC₅₀of the compstatin analog is between about 0.1 μM and about 0.2 μM. Incertain embodiments the IC₅₀ of the compstatin analog is between about0.05 μM and about 0.1 μM. In certain embodiments the IC₅₀ of thecompstatin analog is between about 0.001 μM and about 0.05 μM.

The Kd of compstatin binding to C3 can be measured using isothermaltitration calorimetry (Katragadda, et al., J. Biol. Chem., 279(53),54987-54995, 2004). Binding affinity of a variety of compstatin analogsfor C3 has been correlated with their activity, with a lower K_(d)indicating a higher binding affinity, as recognized in the art. A linearcorrelation between binding affinity and activity was shown for certainanalogs tested (Katragadda, 2004, supra; Katragadda 2006, supra). Incertain embodiments of the invention the compstatin analog binds to C3with a K_(d) of between 0.1 μM and 1.0 μM, between 0.05 μM and 0.1 μM,between 0.025 μM and 0.05 μM, between 0.015 μM and 0.025 μM, between0.01 μM and 0.015 μM, or between 0.001 μM and 0.01 μM.

Compounds “designed or identified based on compstatin” include, but arenot limited to, compounds that comprise an amino acid chain whosesequence is obtained by (i) modifying the sequence of compstatin (e.g.,replacing one or more amino acids of the sequence of compstatin with adifferent amino acid or amino acid analog, inserting one or more aminoacids or amino acid analogs into the sequence of compstatin, or deletingone or more amino acids from the sequence of compstatin); (ii) selectionfrom a phage display peptide library in which one or more amino acids ofcompstatin is randomized, and optionally further modified according tomethod (i); or (iii) identified by screening for compounds that competewith compstatin or any analog thereof obtained by methods (i) or (ii)for binding to C3 or a fragment thereof. Many useful compstatin analogscomprise a hydrophobic cluster, a β-turn, and a disulfide bridge.

In certain embodiments of the invention the sequence of the compstatinanalog comprises or consists essentially of a sequence that is obtainedby making 1, 2, 3, or 4 substitutions in the sequence of compstatin,i.e., 1, 2, 3, or 4 amino acids in the sequence of compstatin isreplaced by a different standard amino acid or by a non-standard aminoacid. In certain embodiments of the invention the amino acid at position4 is altered. In certain embodiments of the invention the amino acid atposition 9 is altered. In certain embodiments of the invention the aminoacids at positions 4 and 9 are altered. In certain embodiments of theinvention only the amino acids at positions 4 and 9 are altered. Incertain embodiments of the invention the amino acid at position 4 or 9is altered, or in certain embodiments both amino acids 4 and 9 arealtered, and in addition up to 2 amino acids located at positionsselected from 1, 7, 10, 11, and 13 are altered. In certain embodimentsof the invention the amino acids at positions 4, 7, and 9 are altered.In certain embodiments of the invention amino acids at position 2, 12,or both are altered, provided that the alteration preserves the abilityof the compound to be cyclized. Such alteration(s) at positions 2 and/or12 may be in addition to the alteration(s) at position 1, 4, 7, 9, 10,11, and/or 13. Optionally the sequence of any of the compstatin analogswhose sequence is obtained by replacing one or more amino acids ofcompstatin sequence further includes up to 1, 2, or 3 additional aminoacids at the C-terminus. In one embodiment, the additional amino acid isGly. Optionally the sequence of any of the compstatin analogs whosesequence is obtained by replacing one or more amino acids of compstatinsequence further includes up to 5, or up to 10 additional amino acids atthe C-terminus. It should be understood that compstatin analogs may haveany one or more of the characteristics or features of the variousembodiments described herein, and characteristics or features of anyembodiment may additionally characterize any other embodiment describedherein, unless otherwise stated or evident from the context. In certainembodiments of the invention the sequence of the compstatin analogcomprises or consists essentially of a sequence identical to that ofcompstatin except at positions corresponding to positions 4 and 9 in thesequence of compstatin.

Compstatin and certain compstatin analogs having somewhat greateractivity than compstatin contain only standard amino acids (“standardamino acids” are glycine, leucine, isoleucine, valine, alanine,phenylalanine, tyrosine, tryptophan, aspartic acid, asparagine, glutamicacid, glutamine, cysteine, methionine, arginine, lysine, proline,serine, threonine and histidine). Certain compstatin analogs havingimproved activity incorporate one or more non-standard amino acids.Useful non-standard amino acids include singly and multiply halogenated(e.g., fluorinated) amino acids, D-amino acids, homo-amino acids,N-alkyl amino acids, dehydroamino acids, aromatic amino acids (otherthan phenylalanine, tyrosine and tryptophan), ortho-, meta- orpara-aminobenzoic acid, phospho-amino acids, methoxylated amino acids,and α,α-disubstituted amino acids. In certain embodiments of theinvention, a compstatin analog is designed by replacing one or moreL-amino acids in a compstatin analog described elsewhere herein with thecorresponding D-amino acid. Such compounds and methods of use thereofare an aspect of the invention. Exemplary non-standard amino acids ofuse include 2-naphthylalanine (2-Nal), 1-naphthylalanine (1-Nal),2-indanylglycine carboxylic acid (2Ig1), dihydrotrpytophan (Dht),4-benzoyl-L-phenylalanine (Bpa), 2-α-aminobutyric acid (2-Abu),3-α-aminobutyric acid (3-Abu), 4-α-aminobutyric acid (4-Abu),cyclohexylalanine (Cha), homocyclohexylalanine (hCha),4-fluoro-L-tryptophan (4fW), 5-fluoro-L-tryptophan (5fW),6-fluoro-L-tryptophan (6fW), 4-hydroxy-L-tryptophan (4OH-W),5-hydroxy-L-tryptophan (5OH-W), 6-hydroxy-L-tryptophan (6OH-W),1-methyl-L-tryptophan (1MeW), 4-methyl-L-tryptophan (4MeW),5-methyl-L-tryptophan (5MeW), 7-aza-L-tryptophan (7aW),α-methyl-L-tryptophan (αMeW), β-methyl-L-tryptophan (βMeW),N-methyl-L-tryptophan (NMeW), ornithine (orn), citrulline, norleucine,γ-glutamic acid, etc.

In certain embodiments of the invention the compstatin analog comprisesone or more Trp analogs (e.g., at position 4 and/or 7 relative to thesequence of compstatin). Exemplary Trp analogs are mentioned above. Seealso Beene, et. al. Biochemistry 41: 10262-10269, 2002 (describing,inter alia, singly- and multiply-halogenated Trp analogs); Babitzke &Yanofsky, J. Biol. Chem. 270: 12452-12456, 1995 (describing, inter alia,methylated and halogenated Trp and other Trp and indole analogs); andU.S. Pat. Nos. 6,214,790, 6,169,057, 5,776,970, 4,870,097, 4,576,750 and4,299,838. Other Trp analogs include variants that are substituted(e.g., by a methyl group) at the α or β carbon and, optionally, also atone or more positions of the indole ring. Amino acids comprising two ormore aromatic rings, including substituted, unsubstituted, oralternatively substituted variants thereof, are of interest as Trpanalogs. In certain embodiments of the invention the Trp analog, e.g.,at position 4, is 5-methoxy, 5-methyl-, 1-methyl-, or1-formyl-tryptophan. In certain embodiments of the invention a Trpanalog (e.g., at position 4) comprising a 1-alkyl substituent, e.g., alower alkyl (e.g., C₁-C₅) substituent is used. In certain embodiments,N(α) methyl tryptophan or 5-methyltryptophan is used. In someembodiments, an analog comprising a 1-alkanyol substituent, e.g., alower alkanoyl (e.g., C₁-C₅) is used. Examples include1-acetyl-L-tryptophan and L-β-tryptophan.

In certain embodiments the Trp analog has increased hydrophobiccharacter relative to Trp. For example, the indole ring may besubstituted by one or more alkyl (e.g., methyl) groups. In certainembodiments the Trp analog participates in a hydrophobic interactionwith C3. Such a Trp analog may be located, e.g., at position 4 relativeto the sequence of compstatin. In certain embodiments the Trp analogcomprises a substituted or unsubstituted bicyclic aromatic ringcomponent or two or more substituted or unsubstituted monocyclicaromatic ring components.

In certain embodiments the Trp analog has increased propensity to formhydrogen bonds with C3 relative to Trp but does not have increasedhydrophobic character relative to Trp. The Trp analog may have increasedpolarity relative to Trp and/or an increased ability to participate inan electrostatic interaction with a hydrogen bond donor on C3. Certainexemplary Trp analogs with an increased hydrogen bond forming charactercomprise an electronegative substituent on the indole ring. Such a Trpanalog may be located, e.g., at position 7 relative to the sequence ofcompstatin.

In certain embodiments of the invention the compstatin analog comprisesone or more Ala analogs (e.g., at position 9 relative to the sequence ofcompstatin), e.g., Ala analogs that are identical to Ala except thatthey include one or more CH₂ groups in the side chain. In certainembodiments the Ala analog is an unbranched single methyl amino acidsuch as 2-Abu. In certain embodiments of the invention the compstatinanalog comprises one or more Trp analogs (e.g., at position 4 and/or 7relative to the sequence of compstatin) and an Ala analog (e.g., atposition 9 relative to the sequence of compstatin).

In certain embodiments of the invention the compstatin analog is acompound that comprises a peptide that has a sequence of

(SEQ ID NO: 2) (X′aa)_(n)-Gln-Asp-Xaa-Gly-(X″aa)_(m),wherein each X′aa and each X″aa is an independently selected amino acidor amino acid analog, wherein Xaa is Trp or an analog of Trp, andwherein n>1 and m>1 and n+m is between 5 and 21. The peptide has a coresequence of

(SEQ ID NO: 70) Gln-Asp-Xaa-Gly,where Xaa is Trp or an analog of Trp, e.g., an analog of Trp havingincreased propensity to form hydrogen bonds with an H-bond donorrelative to Trp but, in certain embodiments, not having increasedhydrophobic character relative to Trp. For example, the analog may beone in which the indole ring of Trp is substituted with anelectronegative moiety, e.g., a halogen such as fluorine. In oneembodiment Xaa is 5-fluorotryptophan. Absent evidence to the contrary,one of skill in the art would recognize that any non-naturally occurringpeptide whose sequence comprises this core sequence and that inhibitscomplement activation and/or binds to C3 will have been designed basedon the sequence of compstatin. In an alternative embodiment Xaa is anamino acid or amino acid analog other than a Trp analog that allows the

(SEQ ID NO: 70) Gln-Asp-Xaa-Glypeptide to form a β-turn.

In certain embodiments of the invention the peptide has a core sequenceof

(SEQ ID NO: 3) X′aa-Gln-Asp-Xaa-Gly,where X′aa and Xaa are selected from Trp and analogs of Trp. In certainembodiments of the invention the peptide has a core sequence of

(SEQ ID NO: 3) X′aa-Gln-Asp-Xaa-Gly,where X′aa and Xaa are selected from Trp, analogs of Trp, and otheramino acids or amino acid analogs comprising at least one aromatic ring.In certain embodiments of the invention the core sequence forms a β-turnin the context of the peptide. The β-turn may be flexible, allowing thepeptide to assume two or more conformations as assessed for example,using nuclear magnetic resonance (NMR). In certain embodiments X′aa isan analog of Trp that comprises a substituted or unsubstituted bicyclicaromatic ring component or two or more substituted or unsubstitutedmonocyclic aromatic ring components. In certain embodiments of theinvention X′aa is selected from the group consisting of2-naphthylalanine, 1-naphthylalanine, 2-indanylglycine carboxylic acid,dihydrotryptophan, and benzoylphenylalanine. In certain embodiments ofthe invention X′aa is an analog of Trp that has increased hydrophobiccharacter relative to Trp. For example, X′aa may be 1-methyltryptophan.In certain embodiments of the invention Xaa is an analog of Trp that hasincreased propensity to form hydrogen bonds relative to Trp but, incertain embodiments, not having increased hydrophobic character relativeto Trp. In certain embodiments of the invention the analog of Trp thathas increased propensity to form hydrogen bonds relative to Trpcomprises a modification on the indole ring of Trp, e.g., at position 5,such as a substitution of a halogen atom for an H atom at position 5.For example, Xaa may be 5-fluorotryptophan.

In certain embodiments of the invention the peptide has a core sequenceof

(SEQ ID NO: 4) X′aa-Gln-Asp-Xaa-Gly-X″aa,where X′aa and Xaa are each independently selected from Trp and analogsof Trp and X″aa is selected from His, Ala, analogs of Ala, Phe, and Trp.In certain embodiments of the invention X′aa is an analog of Trp thathas increased hydrophobic character relative to Trp, such as1-methyltryptophan or another Trp analog having an alkyl substituent onthe indole ring (e.g., at position 1, 4, 5, or 6). In certainembodiments X′aa is an analog of Trp that comprises a substituted orunsubstituted bicyclic aromatic ring component or two or moresubstituted or unsubstituted monocyclic aromatic ring components. Incertain embodiments of the invention X′aa is selected from the groupconsisting of 2-naphthylalanine, 1-naphthylalanine, 2-indanylglycinecarboxylic acid, dihydrotryptophan, and benzoylphenylalanine. In certainembodiments of the invention Xaa is an analog of Trp that has increasedpropensity to form hydrogen bonds with C3 relative to Trp but, incertain embodiments, not having increased hydrophobic character relativeto Trp. In certain embodiments of the invention the analog of Trp thathas increased propensity to form hydrogen bonds relative to Trpcomprises a modification on the indole ring of Trp, e.g., at position 5,such as a substitution of a halogen atom for an H atom at position 5.For example, Xaa may be 5-fluorotryptophan. In certain embodiments X″aais Ala or an analog of Ala such as Abu or another unbranched singlemethyl amino acid. In certain embodiments of the invention the peptidehas a core sequence of

(SEQ ID NO: 4) X′aa-Gln-Asp-Xaa-Gly-X″aa,where X′aa and Xaa are each independently selected from Trp, analogs ofTrp, and amino acids or amino acid analogs comprising at least onearomatic side chain, and X″aa is selected from His, Ala, analogs of Ala,Phe, and Trp. In certain embodiments X″aa is selected from analogs ofTrp, aromatic amino acids, and aromatic amino acid analogs.

In certain preferred embodiments of the invention the peptide is cyclic.The peptide may be cyclized via a bond between any two amino acids, oneof which is (X′aa)_(n) and the other of which is located within(X″aa)_(m). In certain embodiments the cyclic portion of the peptide isbetween 9 and 15 amino acids in length, e.g., 10-12 amino acids inlength. In certain embodiments the cyclic portion of the peptide is 11amino acids in length, with a bond (e.g., a disulfide bond) betweenamino acids at positions 2 and 12. For example, the peptide may be 13amino acids long, with a bond between amino acids at positions 2 and 12resulting in a cyclic portion 11 amino acids in length.

In certain embodiments the peptide comprises or consists of the sequence

(SEQ ID NO: 5) X′aa1-X′aa2-X′aa3-X′aa4-Gln-Asp-Xaa-Gly-X′aa1-X″aa2-X″aa3-X″aa4-X″aa5.In certain embodiments X′aa4 and Xaa are selected from Trp and analogsof Trp, and X′aa1, X′aa2, X′aa3, X″aa1, X″aa2, X″aa3, X″aa4, and X″aa5are independently selected from among amino acids and amino acidanalogs. In certain embodiments X′aa4 and Xaa are selected from aromaticamino acids and aromatic amino acid analogs. Any one or more of X′aa1,X′aa2, X′aa3, X″aa1, X″aa2, X″aa3, X″aa4, and X″aa5 may be identical tothe amino acid at the corresponding position in compstatin. In oneembodiment, X″aa1 is Ala or a single methyl unbranched amino acid. Thepeptide may be cyclized via a covalent bond between (i) X′aa1, X′aa2, orX′aa3; and (ii) X″aa2, X″aa3, X″aa4 or X″aa5. In one embodiment thepeptide is cyclized via a covalent bond between X′aa2 and X″aa4. In oneembodiment the covalently bound amino acid are each Cys and the covalentbond is a disulfide (S—S) bond. In other embodiments the covalent bondis a C—C, C—O, C—S, or C—N bond. In certain embodiments one of thecovalently bound residues is an amino acid or amino acid analog having aside chain that comprises a primary or secondary amine, the othercovalently bound residue is an amino acid or amino acid analog having aside chain that comprises a carboxylic acid group, and the covalent bondis an amide bond. Amino acids or amino acid analogs having a side chainthat comprises a primary or secondary amine include lysine anddiaminocarboxylic acids of general structure NH₂(CH₂)_(n)CH(NH₂)COOHsuch as 2,3-diaminopropionic acid (dapa), 2,4-diaminobutyric acid(daba), and ornithine (orn), wherein n=1 (dapa), 2 (daba), and 3 (orn),respectively. Examples of amino acids having a side chain that comprisesa carboxylic acid group include dicarboxylic amino acids such asglutamic acid and aspartic acid. Analogs such as beta-hydroxy-L-glutamicacid may also be used. In some embodiments a peptide is cyclized with athioether bond, e.g., as described in PCT/US2011/052442(WO/2012/040259). For example, in some embodiments a disulfide bond inany of the peptides is replaced with a thioether bond. In someembodiments, a cystathionine is formed. In some embodiments thecystathionine is a delta-cystathionine or a gamma-cystathionine. In someembodiments a modification comprises replacement of a Cys-Cys disulfidebond between cysteines at X′aa2 and X″aa4 in SEQ ID NO: 5 (orcorresponding positions in other sequences) with addition of a CH₂, toform a homocysteine at X′aa2 or X″aa4, and introduction of a thioetherbond, to form a cystathionine. In one embodiment, the cystathionine is agamma-cystathionine. In another embodiment, the cystathionine is adelta-cystathionine. Another modification in accordance with the presentinvention comprises replacement of the disulfide bond with a thioetherbond without the addition of a CH₂, thereby forming a lantithionine. Insome embodiments a compstatin analog having a thioether in place of adisulfide bond has increased stability, at least under some conditions,as compared with the compstatin analog having the disulfide bond.

In certain embodiments, the compstatin analog is a compound thatcomprises a peptide having a sequence:

(SEQ ID NO: 6) Xaa1-Cys-Val-Xaa2-Gln-Asp-Xaa2*-Gly-Xaa3-His-Arg-Cys-Xaa4;wherein:Xaa1 is Ile, Val, Leu, B¹-Ile, B¹-Val, B¹-Leu or a dipeptide comprisingGly-Ile or B¹-Gly-Ile, and B¹ represents a first blocking moiety;Xaa2 and Xaa2* are independently selected from Trp and analogs of Trp;Xaa3 is His, Ala or an analog of Ala, Phe, Trp, or an analog of Trp;Xaa4 is L-Thr, D-Thr, Ile, Val, Gly, a dipeptide selected from Thr-Alaand Thr-Asn, or a tripeptide comprising Thr-Ala-Asn, wherein a carboxyterminal —OH of any of the L-Thr, D-Thr, Ile, Val, Gly, Ala, or Asnoptionally is replaced by a second blocking moiety B²; and the two Cysresidues are joined by a disulfide bond. In some embodiments, Xaa4 isLeu, Nle, His, or Phe or a dipeptide selected from Xaa5-Ala andXaa5-Asn, or a tripeptide Xaa5-Ala-Asn, wherein Xaa5 is selected fromLeu, Nle, His or Phe, and wherein a carboxy terminal —OH of any of theL-Thr, D-Thr, Ile, Val, Gly, Leu, Nle, His, Phe, Ala, or Asn optionallyis replaced by a second blocking moiety B²; and the two Cys residues arejoined by a disulfide bond.

In other embodiments Xaa1 is absent or is any amino acid or amino acidanalog, and Xaa2, Xaa2*, Xaa3, and Xaa4 are as defined above. If Xaa1 isabsent, the N-terminal Cys residue may have a blocking moiety B¹attached thereto.

In another embodiment, Xaa4 is any amino acid or amino acid analog andXaa1, Xaa2, Xaa2*, and Xaa3 are as defined above. In another embodimentXaa4 is a dipeptide selected from the group consisting of: Thr-Ala andThr-Asn, wherein the carboxy terminal —OH or the Ala or Asn isoptionally replaced by a second blocking moiety B².

In any of the embodiments of the compstatin analog of SEQ ID NO: 6, Xaa2may be Trp.

In any of the embodiments of the compstatin analog of SEQ ID NO: 6, Xaa2may be an analog of Trp comprising a substituted or unsubstitutedbicyclic aromatic ring component or two or more substituted orunsubstituted monocyclic aromatic ring components. For example, theanalog of Trp may be selected from 2-naphthylalanine (2-Nal),1-naphthylalanine (1-Nal), 2-indanylglycine carboxylic acid (Ig1),dihydrotrpytophan (Dht), and 4-benzoyl-L-phenylalanine.

In any of the embodiments of the compstatin analog of SEQ ID NO: 6, Xaa2may be an analog of Trp having increased hydrophobic character relativeto Trp. For example, the analog of Trp may be selected from1-methyltryptophan, 4-methyltryptophan, 5-methyltryptophan, and6-methyltryptophan. In one embodiment, the analog of Trp is1-methyltryptophan. In one embodiment, Xaa2 is 1-methyltryptophan, Xaa2*is Trp, Xaa3 is Ala, and the other amino acids are identical to those ofcompstatin.

In any of the embodiments of the compstatin analog of SEQ ID NO: 6,Xaa2* may be an analog of Trp such as an analog of Trp having increasedhydrogen bond forming propensity with C3 relative to Trp, which, incertain embodiments, does not have increased hydrophobic characterrelative to Trp. In certain embodiments the analog of Trp comprises anelectronegative substituent on the indole ring. For example, the analogof Trp may be selected from 5-fluorotryptophan and 6-fluorotryptophan.

In certain embodiments of the invention Xaa2 is Trp and Xaa2* is ananalog of Trp having increased hydrogen bond forming propensity with C3relative to Trp which, in certain embodiments, does not have increasedhydrophobic character relative to Trp. In certain embodiments of thecompstatin analog of SEQ ID NO: 6, Xaa2 is analog of Trp havingincreased hydrophobic character relative to Trp such as an analog of Trpselected from 1-methyltryptophan, 4-methyltryptophan,5-methyltryptophan, and 6-methyltryptophan, and Xaa2* is an analog ofTrp having increased hydrogen bond forming propensity with C3 relativeto Trp which, in certain embodiments, does not have increasedhydrophobic character relative to Trp. For example, in one embodimentXaa2 is methyltryptophan and Xaa2* is 5-fluorotryptophan.

In certain of the afore-mentioned embodiments, Xaa3 is Ala. In certainof the afore-mentioned embodiments Xaa3 is a single methyl unbranchedamino acid, e.g., Abu.

The invention further provides compstatin analogs of SEQ ID NO: 6, asdescribed above, wherein Xaa2 and Xaa2* are independently selected fromTrp, analogs of Trp, and other amino acids or amino acid analogs thatcomprise at least one aromatic ring, and Xaa3 is His, Ala or an analogof Ala, Phe, Trp, an analog of Trp, or another aromatic amino acid oraromatic amino acid analog.

In certain embodiments of the invention the blocking moiety present atthe N- or C-terminus of any of the compstatin analogs described hereinis any moiety that stabilizes a peptide against degradation that wouldotherwise occur in mammalian (e.g., human or non-human primate) blood orinterstitial fluid. For example, blocking moiety B¹ could be any moietythat alters the structure of the N-terminus of a peptide so as toinhibit cleavage of a peptide bond between the N-terminal amino acid ofthe peptide and the adjacent amino acid. Blocking moiety B² could be anymoiety that alters the structure of the C-terminus of a peptide so as toinhibit cleavage of a peptide bond between the C-terminal amino acid ofthe peptide and the adjacent amino acid. Any suitable blocking moietiesknown in the art could be used. In certain embodiments of the inventionblocking moiety B¹ comprises an acyl group (i.e., the portion of acarboxylic acid that remains following removal of the —OH group). Theacyl group typically comprises between 1 and 12 carbons, e.g., between 1and 6 carbons. For example, in certain embodiments of the inventionblocking moiety B¹ is selected from the group consisting of: formyl,acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, etc. In oneembodiment, the blocking moiety B¹ is an acetyl group, i.e., Xaa1 isAc-Ile, Ac-Val, Ac-Leu, or Ac-Gly-Ile.

In certain embodiments of the invention blocking moiety B² is a primaryor secondary amine (—NH₂ or —NHR¹, wherein R is an organic moiety suchas an alkyl group).

In certain embodiments of the invention blocking moiety B¹ is any moietythat neutralizes or reduces the positive charge that may otherwise bepresent at the N-terminus at physiological pH. In certain embodiments ofthe invention blocking moiety B² is any moiety that neutralizes orreduces the negative charge that may otherwise be present at theC-terminus at physiological pH.

In certain embodiments of the invention, the compstatin analog isacetylated or amidated at the N-terminus and/or C-terminus,respectively. A compstatin analog may be acetylated at the N-terminus,amidated at the C-terminus, and or both acetylated at the N-terminus andamidated at the C-terminus. In certain embodiments of the invention acompstatin analog comprises an alkyl or aryl group at the N-terminusrather than an acetyl group.

In certain embodiments, the compstatin analog is a compound thatcomprises a peptide having a sequence:

(SEQ ID NO: 7) Xaa1-Cys-Val-Xaa2-Gln-Asp-Xaa2*-Gly-Xaa3-His-Arg-Cys-Xaa4;wherein:Xaa1 is Ile, Val, Leu, Ac-Ile, Ac-Val, Ac-Leu or a dipeptide comprisingGly-Ile or Ac-Gly-Ile;Xaa2 and Xaa2* are independently selected from Trp and analogs of Trp;Xaa3 is His, Ala or an analog of Ala, Phe, Trp, or an analog of Trp;Xaa4 is L-Thr, D-Thr, Ile, Val, Gly, a dipeptide selected from Thr-Alaand Thr-Asn, or a tripeptide comprising Thr-Ala-Asn, wherein a carboxyterminal —OH of any of L-Thr, D-Thr, Ile, Val, Gly, Ala, or Asnoptionally is replaced by —NH₂; and the two Cys residues are joined by adisulfide bond. In some embodiments, Xaa4 is Leu, Nle, His, or Phe or adipeptide selected from Xaa5-Ala and Xaa5-Asn, or a tripeptideXaa5-Ala-Asn, wherein Xaa5 is selected from Leu, Nle, His or Phe, andwherein a carboxy terminal —OH of any of the L-Thr, D-Thr, Ile, Val,Gly, Leu, Nle, His, Phe, Ala, or Asn optionally is replaced by a secondblocking moiety B2; and the two Cys residues are joined by a disulfidebond.

In some embodiments, Xaa1, Xaa2, Xaa2*, Xaa3, and Xaa4 are as describedabove for the various embodiments of SEQ ID NO: 6. For example, incertain embodiments Xaa2* is Trp. In certain embodiments Xaa2 is ananalog of Trp having increased hydrophobic character relative to Trp,e.g., 1-methyltryptophan. In certain embodiments Xaa3 is Ala. In certainembodiments Xaa3 is a single methyl unbranched amino acid.

In certain embodiments of the invention Xaa1 is Ile and Xaa4 is L-Thr.

In certain embodiments of the invention Xaa1 is Ile, Xaa2* is Trp, andXaa4 is L-Thr.

The invention further provides compstatin analogs of SEQ ID NO: 7, asdescribed above, wherein Xaa2 and Xaa2* are independently selected fromTrp, analogs of Trp, other amino acids or aromatic amino acid analogs,and Xaa3 is His, Ala or an analog of Ala, Phe, Trp, an analog of Trp, oranother aromatic amino acid or aromatic amino acid analog.

In certain embodiments of any of the compstatin analogs describedherein, an analog of Phe is used rather than Phe.

Table 1 provides a non-limiting list of compstatin analogs useful in thepresent invention. The analogs are referred to in abbreviated form inthe left column by indicating specific modifications at designatedpositions (1-13) as compared to the parent peptide, compstatin.Consistent with usage in the art, “compstatin” as used herein, and theactivities of compstatin analogs described herein relative to that ofcompstatin, refer to the compstatin peptide amidated at the C-terminus.Unless otherwise indicated, peptides in Table 1 are amidated at theC-terminus. Bold text is used to indicate certain modifications.Activity relative to compstatin is based on published data and assaysdescribed therein (WO2004/026328, WO2007044668, Mallik, 2005;Katragadda, 2006). Where multiple publications reporting an activitywere consulted, the more recently published value is used, and it willbe recognized that values may be adjusted in the case of differencesbetween assays. It will also be appreciated that in certain embodimentsof the invention the peptides listed in Table 1 are cyclized via adisulfide bond between the two Cys residues when used in the therapeuticcompositions and methods of the invention. Alternate means for cyclizingthe peptides are also within the scope of the invention. As noted above,in various embodiments of the invention one or more amino acid(s) of acompstatin analog (e.g., any of the compstatin analogs disclosed herein)can be an N-alkyl amino acid (e.g., an N-methyl amino acid). Forexample, and without limitation, at least one amino acid within thecyclic portion of the peptide, at least one amino acid N-terminal to thecyclic portion, and/or at least one amino acid C-terminal to the cyclicportion may be an N-alkyl amino acid, e.g., an N-methyl amino acid. Insome embodiments of the invention, for example, a compstatin analogcomprises an N-methyl glycine, e.g., at the position corresponding toposition 8 of compstatin and/or at the position corresponding toposition 13 of compstatin. In some embodiments, one or more of thecompstatin analogs in Table 1 contains at least one N-methyl glycine,e.g., at the position corresponding to position 8 of compstatin and/orat the position corresponding to position 13 of compstatin. In someembodiments, one or more of the compstatin analogs in Table 1 containsat least one N-methyl isoleucine, e.g., at the position corresponding toposition 13 of compstatin. For example, a Thr at or near the C-terminalend of a peptide whose sequence is listed in Table 1 or any othercompstatin analog sequence may be replaced by N-methyl Ile. As will beappreciated, in some embodiments the N-methylated amino acids compriseN-methyl Gly at position 8 and N-methyl Ile at position 13. In someembodiments the N-methylated amino acids comprise N-methyl Gly in a coresequence such as SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments theN-methylated amino acids comprise N-methyl Gly in a core sequence suchas SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.

TABLE 1 SEQ ID Activity over Peptide Sequence NO: compstatin CompstatinH-ICVVQDWGHHRCT-CONH2 8 * Ac-compstatin Ac-ICVVQDWGHHRCT-CONH2 9  3×more Ac-V4Y/H9A Ac-ICV Y QDWG A HRCT-CONH2 10 14× more Ac-V4W/H9A-OHAc-ICV W QDWG A HRCT-COOH 11 27× more Ac-V4W/H9A Ac-ICV W QDWG AHRCT-CONH2 12 45× more Ac-V4W/H9A/T13dT-OH Ac-ICV W QDWG A HRC dT -COOH13 55× more Ac-V4(2-Nal)/H9A Ac-ICV (2-Nal) QDWG A HRCT-CONH2 14 99×more Ac V4(2-Nal)/H9A-OH Ac-ICV (2-Nal) QDWG A HRCT-COOH 15 38× moreAc V4(1-Nal)/H9A-OH Ac-ICV (1-Nal) QDWG A HRCT-COOH 16 30× moreAc-V42IgI/H9A Ac-ICV(2- IgI) QDWG A HRCT-CONH2 17 39× moreAc-V42IgI/H9A-OH Ac-ICV(2- IgI) QDWG A HRCT-COOH 18 37× moreAc-V4Dht/H9A-OH Ac-ICV Dht QDWG A HRCT-COOH 19  5× moreAc-V4(Bpa)/H9A-OH Ac-ICV (Bpa) QDWG A HRCT-COOH 20 49× moreAc-V4(Bpa)/H9A Ac-ICV (Bpa) QDWG A HRCT-CONH2 21 86× moreAc-V4(Bta)/H9A-OH Ac-ICV (Bta) QDWG A HRCT-COOH 22 65× moreAc-V4(Bta)/H9A Ac-ICV (Bta) QDWG A HRCT-CONH2 23 64× moreAc-V4W/H9(2-Abu) Ac-ICV W QDWG(2- Abu) HRCT-CONH2 24 64× more+G/V4W/H9A + AN-OH H- G ICV W QDWG A HRCTA N -COOH 25 38× moreAc-V4(5fW)/H9A Ac-ICV (5fW) QDWG A HRCT-CONH₂ 26 31× moreAc-V4(5-MeW)/H9A Ac-ICV (5-methyl-W) QDWG A HRCT-CONH₂ 27 67× moreAc-V4(1-MeW)/H9A Ac-ICV (1-methyl-W) QDWG A HRCT-CONH₂ 28 264× more Ac-V4W/W7(5fW)/H9A Ac-ICV W QD( 5fW) G A HRCT-CONH₂ 29 121× more Ac-V4(5fW)/W7(5fW)/H9A Ac-ICV (5fW) QD( 5fW) G A HRCT-CONH₂ 30 NAAc-V4(5-MeW)/W7(5fW)H9A Ac-ICV (5-methyl-W) QD( 5fW) G A HRCT- 31 NACONH₂ Ac-V4 (1MeW)/W7(5fW)/H9A Ac-ICV(1-methyl-W)QD(5fW)G A HRCT- 32264× more  CONH₂ +G/V4(6fW)/W7(6fW)H9A + N- H-GICV (6fW )QD(6fW)G A HRCTN -COOH 33 126× more  OH Ac-V4(1-formyl-W)/H9A Ac-ICV( 1-formyl-W )QDWGA HRCT-CONH₂ 34 264× more  Ac-V4(5-methoxy-W)/H9A Ac-ICV (1-methyoxy-W)QDWG A HRCT- 35 76× more CONH₂ G/V4(5f-W)/W7(5fW)/H9A + N- H-GICV( 5fW)QD( 5fW )GAHRCT N -COOH 36 112× more  OH NA = not available

In certain embodiments of the compositions and methods of the inventionthe compstatin analog has a sequence selected from sequences 9-36. Incertain embodiments of the compositions and methods of the invention thecompstatin analog has a sequence selected from SEQ ID NOs: 14, 21, 28,29, 32, 33, 34, and 36. In certain embodiments of the compositionsand/or methods of the invention the compstatin analog has a sequenceselected from SEQ ID NOs: 30 and 31. In one embodiment of thecompositions and methods of the invention the compstatin analog has asequence of SEQ ID NO: 28. In one embodiment of the compositions andmethods of the invention the compstatin analog has a sequence of SEQ IDNO: 32. In one embodiment of the compositions and methods of theinvention the compstatin analog has a sequence of SEQ ID NO: 34. In oneembodiment of the compositions and methods of the invention thecompstatin analog has a sequence of SEQ ID NO: 36.

In some embodiments a blocking moiety B¹ comprises an amino acid, whichmay be represented as Xaa0. In some embodiments blocking moiety B²comprises an amino acid, which may be represented as XaaN. In someembodiments blocking moiety B¹ and/or B² comprises a non-standard aminoacid, such as a D-amino acid, N-alkyl amino acid (e.g., N-methyl aminoacid). In some embodiments a blocking moiety B¹ and/or B² comprises anon-standard amino acid that is an analog of a standard amino acid. Insome embodiments an amino acid analog comprises a lower alkyl, loweralkoxy, or halogen substituent, as compared with a standard amino acidof which it is an analog. In some embodiments a substituent is on a sidechain. In some embodiments a substituent is on an alpha carbon atom. Insome embodiments, a blocking moiety B¹ comprising an amino acid, e.g., anon-standard amino acid, further comprises a moiety B^(1a). For example,blocking moiety B¹ may be represented as B^(1a)-Xaa0. In someembodiments B^(1a) neutralizes or reduces a positive charge that mayotherwise be present at the N-terminus at physiological pH. In someembodiments B^(1a) comprises or consists of, e.g., an acyl group that,e.g., comprises between 1 and 12 carbons, e.g., between 1 and 6 carbons.In certain embodiments blocking moiety B^(1a) is selected from the groupconsisting of: formyl, acetyl, proprionyl, butyryl, isobutyryl, valeryl,isovaleryl, etc. In some embodiments, a blocking moiety B² comprising anamino acid, e.g., a non-standard amino acid, may further comprise amoiety B^(2a) For example, blocking moiety B² may be represented asXaaN-B^(2a), where N represents the appropriate number for the aminoacid (which will depend on the numbering used in the rest of thepeptide). In some embodiments B^(2a) neutralizes or reduces a negativecharge that may otherwise be present at the C-terminus at physiologicalpH. In some embodiments B^(2a) comprises or consists of a primary orsecondary amine (e.g., NH₂). It will be understood that a blockingactivity of moiety B^(1a)-Xaa0 and/or XaaN-B^(2a) may be provided byeither or both components of the moiety in various embodiments. In someembodiments a blocking moiety or portion thereof, e.g., an amino acidresidue, may contribute to increasing affinity of the compound for C3 orC3b and/or improve the activity of the compound. In some embodiments acontribution to affinity or activity of an amino acid residue may be atleast as important as a contribution to blocking activity. For example,in some embodiments Xaa0 and/or XaaN in B^(1a)-Xaa0 and/or XaaN-B^(2a)may function mainly to increase affinity or activity of the compound,while B^(1a) and/or B^(2a) may inhibit digestion of and/or neutralize acharge of the peptide. In some embodiments a compstatin analog comprisesthe amino acid sequence of any of SEQ ID NOs: 5-36, wherein SEQ ID NOs:5-36 is further extended at the N- and/or C-terminus. In someembodiments, the sequence may be represented asB^(1a)-Xaa0-SEQUENCE-XaaN-B^(2a), where SEQUENCE represents any of SEQID NOs: 5-36, wherein B^(1a) and B^(2a) may independently be present orabsent. For example, in some embodiments a compstatin analog comprises

(SEQ ID NO: 71) B^(1a)-Xaa0-X′aa1-X′aa2-X′aa3-X′aa4-Gln-Asp-Xaa-Gly-X″aa1-X″aa2-X″aa3-X″aa4-X″aa5-XaaN-B^(2a),where X′aa1-X′aa2-X′aa3-X′aa4, Xaa, X″aa1, X″aa2, X″aa3, X″aa4, andX″aa5 are as set forth above for SEQ ID NO: 5.

In some embodiments a compstatin analog comprises

(SEQ ID NO: 72) B^(1a)-Xaa0-Xaa1-Cys-Val-Xaa2-Gln-Asp-Xaa2*-Gly-Xaa3-His-Arg-Cys-Xaa4-XaaN-B^(2a),where Xaa1, Xaa2, Xaa2*, Xaa3, and Xaa4 are as set forth above for SEQID NO: 6 or wherein Xaa1, Xaa2, Xaa2*, Xaa3, and Xaa4 are as set forthfor SEQ ID NO: 6 or SEQ ID NO: 7.

In some embodiments a compstatin analog comprises

(SEQ ID NO: 73) B^(1a)-Xaa0-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Xaa11-Xaa12-Xaa13-XaaN-B^(2a)wherein Xaa1, Xaa2, Xaa3, Xaa4, Xaa5, Xaa6, Xaa7, Xaa8, Xaa9, Xaa10,Xaa11, Xaa12, and Xaa13 are identical to amino acids at positions 1-13of any of SEQ ID NOs: 9-36.

In some embodiments Xaa0 and/or XaaN in any compstatin analog sequencecomprises an amino acid that comprises an aromatic ring having an alkylsubstituent at one or more positions. In some embodiments an alkylsubstituent is a lower alkyl substituent. For example, in someembodiments an alkyl substituent is a methyl or ethyl group. In someembodiments a substituent is located at any position that does notdestroy the aromatic character of the compound. In some embodiments asubstituent is located at any position that does not destroy thearomatic character of a ring to which the substituent is attached. Insome embodiments a substituent is located at position 1, 2, 3, 4, or 5.In some embodiments Xaa0 comprises an O-methyl analog of tyrosine,2-hydroxyphenylalanine or 3-hydroxyphenylalanine. For purposes of thepresent disclosure, a lower case “m” followed by a three letter aminoacid abbreviation may be used to specifically indicate that the aminoacid is an N-methyl amino acid. For example, where the abbreviation“mGly” appears herein, it denotes N-methyl glycine (also sometimesreferred to as sarcosine or Sar). In some embodiments Xaa0 is orcomprises mGly, Tyr, Phe, Arg, Trp, Thr, Tyr(Me), Cha, mPhe, mVal, mile,mAla, DTyr, DPhe, DArg, DTrp, DThr, DTyr(Me), mPhe, mVal, mile, DAla, orDCha. For example, in some embodiments a compstatin analog comprises apeptide having a sequence

(SEQ ID NO: 74) B¹-Ile-[Cys-Val-Trp(Me)-Gln-Asp-Trp-mGly-Ala-His-Arg-Cys]-mIle-B²The two Cys residues are joined by a disulfide bond in the activecompounds. In some embodiments the peptide is acetylated at theN-terminus and/or amidated at the C-terminus. In some embodiments B¹comprises B^(1a)-Xaa0 and/or B² comprises XaaN-B^(2a), as describedabove. For example, in some embodiments B¹ comprises or consists of Gly,mGly, Tyr, Phe, Arg, Trp, Thr, Tyr(Me), mPhe, mVal, mile, mAla, DTyr,DPhe, DTrp, DCha, DAla and B² comprises NH₂, e.g., a carboxy terminal—OH of mile is replaced by NH₂. In some embodiments B¹ comprises orconsists of mGly, Tyr, DTyr, or Tyr(Me) and B² comprises NH₂, e.g., acarboxy terminal —OH of mile is replaced by NH₂. In some embodiments anIle at position Xaa1 is replaced by Gly. Complement inhibition potencyand/or C3b binding parameters of selected compstatin analogs aredescribed in WO/2010/127336 (PCT/US2010/033345) and/or in Qu, et al.,Immunobiology (2012), doi:10.1016/j.imbio.2012.06.003.

In some embodiments a blocking moiety or portion thereof, e.g., an aminoacid residue, may contribute to increasing affinity of the compound forC3 or C3b and/or improve the activity of the compound. In someembodiments a contribution to affinity or activity of an amino acid oramino acid analog may be more significant than a blocking activity.

In certain embodiments of the compositions and methods of the inventionthe compstatin analog has a sequence as set forth in Table 1, but wherethe Ac— group is replaced by an alternate blocking moiety B¹, asdescribed herein. In some embodiments the —NH₂ group is replaced by analternate blocking moiety B², as described herein.

In one embodiment, the compstatin analog binds to substantially the sameregion of the β chain of human C3 as does compstatin. In one embodimentthe compstatin analog is a compound that binds to a fragment of theC-terminal portion of the β chain of human C3 having a molecular weightof about 40 kDa to which compstatin binds (Soulika, A. M., et al., Mol.Immunol., 35:160, 1998; Soulika, A. M., et al., Mol. Immunol.43(12):2023-9, 2006). In certain embodiments the compstatin analog is acompound that binds to the binding site of compstatin as determined in acompstatin-C3 structure, e.g., a crystal structure or NMR-derived 3Dstructure. In certain embodiments the compstatin analog is a compoundthat could substitute for compstatin in a compstatin-C3 structure andwould form substantially the same intermolecular contacts with C3 ascompstatin. In certain embodiments the compstatin analog is a compoundthat binds to the binding site of a peptide having a sequence set forthin Table 1, e.g., SEQ ID NO: 14, 21, 28, 29, 32, 33, 34, or 36, 37, 71,72, 73, or 74 or another compstatin analog sequence disclosed herein ina peptide-C3 structure, e.g., a crystal structure. In certainembodiments the compstatin analog is a compound that binds to thebinding site of a peptide having SEQ ID NO: 30 or 31 in a peptide-C3structure, e.g., a crystal structure. In certain embodiments thecompstatin analog is a compound that could substitute for the peptide ofSEQ ID NO: 9-36, e.g., a compound that could substitute for the peptideof SEQ ID NO: 14, 21, 28, 29, 32, 33, 34, or 36, 37, 71, 72, 73, or 74or another compstatin analog sequence disclosed herein in a peptide-C3structure and would form substantially the same intermolecular contactswith C3 as the peptide. In certain embodiments the compstatin analog isa compound that could substitute for the peptide of SEQ ID NO: 30 or 31in a peptide-C3 structure and would form substantially the sameintermolecular contacts with C3 as the peptide.

One of ordinary skill in the art will readily be able to determinewhether a compstatin analog binds to a fragment of the C-terminalportion of the β chain of C3 using routine experimental methods. Forexample, one of skill in the art could synthesize a photocrosslinkableversion of the compstatin analog by including a photo-crosslinking aminoacid such as p-benzoyl-L-phenylalanine (Bpa) in the compound, e.g., atthe C-terminus of the sequence (Soulika, A. M., et al, supra).Optionally additional amino acids, e.g., an epitope tag such as a FLAGtag or an HA tag could be included to facilitate detection of thecompound, e.g., by Western blotting. The compstatin analog is incubatedwith the fragment and crosslinking is initiated. Colocalization of thecompstatin analog and the C3 fragment indicates binding. Surface plasmonresonance may also be used to determine whether a compstatin analogbinds to the compstatin binding site on C3 or a fragment thereof. One ofskill in the art would be able to use molecular modeling softwareprograms to predict whether a compound would form substantially the sameintermolecular contacts with C3 as would compstatin or a peptide havingthe sequence of any of the peptides in Table 1, e.g., SEQ ID NO: 14, 21,28, 29, 32, 33, 34, or 36, or in some embodiments SEQ ID NO: 30 or 31,37, 71, 72, 73, or 74 or another compstatin analog sequence disclosedherein.

Compstatin analogs may be prepared by various synthetic methods ofpeptide synthesis known in the art via condensation of amino acidresidues, e.g., in accordance with conventional peptide synthesismethods, may be prepared by expression in vitro or in living cells fromappropriate nucleic acid sequences encoding them using methods known inthe art. For example, peptides may be synthesized using standardsolid-phase methodologies as described in Malik, supra, Katragadda,supra, WO2004026328, and/or WO2007062249. Potentially reactive moietiessuch as amino and carboxyl groups, reactive functional groups, etc., maybe protected and subsequently deprotected using various protectinggroups and methodologies known in the art. See, e.g., “Protective Groupsin Organic Synthesis”, 3^(rd) ed. Greene, T. W. and Wuts, P. G., Eds.,John Wiley & Sons, New York: 1999. Peptides may be purified usingstandard approaches such as reversed-phase HPLC. Separation ofdiasteriomeric peptides, if desired, may be performed using knownmethods such as reversed-phase HPLC. Preparations may be lyophilized, ifdesired, and subsequently dissolved in a suitable solvent, e.g., water.The pH of the resulting solution may be adjusted, e.g. to physiologicalpH, using a base such as NaOH. Peptide preparations may be characterizedby mass spectrometry if desired, e.g., to confirm mass and/or disulfidebond formation. See, e.g., Mallik, 2005, and Katragadda, 2006.

A compstatin analog, optionally linked to a cell-reactive moiety ortargeting moiety, can be modified by addition of a molecule such aspolyethylene glycol (PEG) or similar molecules to stabilize thecompound, reduce its immunogenicity, increase its lifetime in the body,increase or decrease its solubility, and/or increase its resistance todegradation. Methods for pegylation are well known in the art (Veronese,F. M. & Harris, Adv. Drug Deliv. Rev. 54, 453-456, 2002; Davis, F. F.,Adv. Drug Deliv. Rev. 54, 457-458, 2002); Hinds, K. D. & Kim, S. W. Adv.Drug Deliv. Rev. 54, 505-530 (2002; Roberts, M. J., Bentley, M. D. &Harris, J. M. Adv. Drug Deliv. Rev. 54, 459-476; 2002); Wang, Y. S. etal. Adv. Drug Deliv. Rev. 54, 547-570, 2002). A wide variety of polymerssuch as PEGs and modified PEGs, including derivatized PEGs to whichpolypeptides can conveniently be attached are described in NektarAdvanced Pegylation 2005-2006 Product Catalog, Nektar Therapeutics, SanCarlos, Calif., which also provides details of appropriate conjugationprocedures. In another embodiment a compstatin analog is fused to the Fcdomain of an immunoglobulin or a portion thereof. In some otherembodiments a compstatin analog is conjugated to an albumin moiety or toan albumin binding peptide. Thus in some embodiments a compstatin analogis modified with one or more polypeptide or non-polypeptide components,e.g., the compstatin analog is pegylated or conjugated to anothermoiety. In some embodiments the component is not the Fc domain of animmunoglobulin or a portion thereof. A compstatin analog can be providedas a multimer or as part of a supramolecular complex, which can includeeither a single molecular species or multiple different species (e.g.,multiple different analogs).

In some embodiments, a compstatin analog is a multivalent compoundcomprising a plurality of compstatin analog moieties covalently ornoncovalently linked to a polymeric backbone or scaffold. The compstatinanalog moieties can be identical or different. In certain embodiments ofthe invention the multivalent compound comprises multiple instances, orcopies, of a single compstatin analog moiety. In other embodiments ofthe invention the multivalent compound comprises one or more instancesof each of two of more non-identical compstatin analog moieties, e.g.,3, 4, 5, or more different compstatin analog moieties. In certainembodiments of the invention the number of compstatin analog moieties(“n”) is between 2 and 6. In other embodiments of the invention n isbetween 7 and 20. In other embodiments of the invention n is between 20and 100. In other embodiments n is between 100 and 1,000. In otherembodiments of the invention n is between 1,000 and 10,000. In otherembodiments n is between 10,000 and 50,000. In other embodiments n isbetween 50,000 and 100,000. In other embodiments n is between 100,000and 1,000,000.

The compstatin analog moieties may be attached directly to the polymericscaffold or may be attached via a linking moiety that connects thecompstatin analog moiety to the polymeric scaffold. The linking moietymay be attached to a single compstatin analog moiety and to thepolymeric scaffold. Alternately, a linking moiety may have multiplecompstatin analog moieties joined thereto so that the linking moietyattaches multiple compstatin analog moieties to the polymeric scaffold.

In some embodiments, the compstatin analog comprises an amino acidhaving a side chain comprising a primary or secondary amine, e.g., a Lysresidue. For example, a Lys residue, or a sequence comprising a Lysresidue, is added at the N-terminus and/or C-terminus of the compstatinanalog. In some embodiments, the Lys residue is separated from thecyclic portion of the compstatin analog by a rigid or flexible spacer.The spacer may, for example, comprise a substituted or unsubstituted,saturated or unsaturated alkyl chain, oligo(ethylene glycol) chain,and/or other moieties, e.g., as described in Section VI with regard tolinkers. The length of the chain may be, e.g., between 2 and 20 carbonatoms. In other embodiments the spacer is a peptide. The peptide spacermay be, e.g., between 1 and 20 amino acids in length, e.g., between 4and 20 amino acids in length. Suitable spacers can comprise or consistof multiple Gly residues, Ser residues, or both, for example.Optionally, the amino acid having a side chain comprising a primary orsecondary amine and/or at least one amino acid in a spacer is a D-aminoacid. Any of a variety of polymeric backbones or scaffolds could beused. For example, the polymeric backbone or scaffold may be apolyamide, polysaccharide, polyanhydride, polyacrylamide,polymethacrylate, polypeptide, polyethylene oxide, or dendrimer.Suitable methods and polymeric backbones are described, e.g., inWO98/46270 (PCT/US98/07171) or WO98/47002 (PCT/US98/06963). In oneembodiment, the polymeric backbone or scaffold comprises multiplereactive functional groups, such as carboxylic acids, anhydride, orsuccinimide groups. The polymeric backbone or scaffold is reacted withthe compstatin analogs. In one embodiment, the compstatin analogcomprises any of a number of different reactive functional groups, suchas carboxylic acids, anhydride, or succinimide groups, which are reactedwith appropriate groups on the polymeric backbone. Alternately,monomeric units that could be joined to one another to form a polymericbackbone or scaffold are first reacted with the compstatin analogs andthe resulting monomers are polymerized. In another embodiment, shortchains are prepolymerized, functionalized, and then a mixture of shortchains of different composition are assembled into longer polymers.

V. Compstatin Mimetics

The structure of compstatin is known in the art, and NMR structures fora number of compstatin analogs having higher activity than compstatinare also known (Malik, supra). Structural information may be used todesign compstatin mimetics.

In one embodiment, the compstatin mimetic is any compound that competeswith compstatin or any compstatin analog (e.g., a compstatin analogwhose sequence is set forth in Table 1) for binding to C3 or a fragmentthereof (such as a 40 kD fragment of the β chain to which compstatinbinds). In some embodiments, the compstatin mimetic has an activityequal to or greater than that of compstatin. In some embodiments, thecompstatin mimetic is more stable, orally available, or has a betterbioavailability than compstatin. The compstatin mimetic may be apeptide, nucleic acid, or small molecule. In certain embodiments thecompstatin mimetic is a compound that binds to the binding site ofcompstatin as determined in a compstatin-C3 structure, e.g., a crystalstructure or a 3-D structure derived from NMR experiments. In certainembodiments the compstatin mimetic is a compound that could substitutefor compstatin in a compstatin-C3 structure and would form substantiallythe same intermolecular contacts with C3 as compstatin. In certainembodiments the compstatin mimetic is a compound that binds to thebinding site of a peptide having a sequence set forth in Table 1, e.g.,SEQ ID NO: 14, 21, 28, 29, 32, 33, 34, or 36 or in certain embodimentsSEQ ID NO: 30 or 31 or other compstatin analog sequence, in a peptide-C3structure. In certain embodiments the compstatin mimetic is a compoundthat could substitute for a peptide having a sequence set forth in Table1, e.g., SEQ ID NO: 14, 21, 28, 29, 32, 33, 34, or 36 or in certainembodiments SEQ ID NO: 30 or 31 or other compstatin analog sequence, ina peptide-C3 structure and would form substantially the sameintermolecular contacts with C3 as the peptide. In certain embodimentsthe compstatin mimetic has a non-peptide backbone but has side chainsarranged in a sequence designed based on the sequence of compstatin.

One of skill in the art will appreciate that once a particular desiredconformation of a short peptide has been ascertained, methods fordesigning a peptide or peptidomimetic to fit that conformation are wellknown. See, e.g., G. R. Marshall (1993), Tetrahedron, 49: 3547-3558;Hruby and Nikiforovich (1991), in Molecular Conformation and BiologicalInteractions, P. Balaram & S. Ramasehan, eds., Indian Acad. of Sci.,Bangalore, PP. 429-455), Eguchi M, Kahn M., Mini Rev Med Chem.,2(5):447-62, 2002. Of particular relevance to the present invention, thedesign of peptide analogs may be further refined by considering thecontribution of various side chains of amino acid residues, e.g., forthe effect of functional groups or for steric considerations asdescribed in the art for compstatin and analogs thereof, among others.

It will be appreciated by those of skill in the art that a peptide mimicmay serve equally well as a peptide for the purpose of providing thespecific backbone conformation and side chain functionalities requiredfor binding to C3 and inhibiting complement activation. Accordingly, itis contemplated as being within the scope of the present invention toproduce and utilize C3-binding, complement-inhibiting compounds throughthe use of either naturally-occurring amino acids, amino acidderivatives, analogs or non-amino acid molecules capable of being joinedto form the appropriate backbone conformation. A non-peptide analog, oran analog comprising peptide and non-peptide components, is sometimesreferred to herein as a “peptidomimetic” or “isosteric mimetic,” todesignate substitutions or derivations of a peptide that possesses muchthe same backbone conformational features and/or other functionalities,so as to be sufficiently similar to the exemplified peptides to inhibitcomplement activation. More generally, a compstatin mimetic is anycompound that would position pharmacophores similarly to theirpositioning in compstatin, even if the backbone differs.

The use of peptidomimetics for the development of high-affinity peptideanalogs is well known in the art. Assuming rotational constraintssimilar to those of amino acid residues within a peptide, analogscomprising non-amino acid moieties may be analyzed, and theirconformational motifs verified, by means of the Ramachandran plot (Hruby& Nikiforovich 1991), among other known techniques.

One of skill in the art will readily be able to establish suitablescreening assays to identify additional compstatin mimetics and toselect those having desired inhibitory activities. For example,compstatin or an analog thereof could be labeled (e.g., with aradioactive or fluorescent label) and contacted with C3 in the presenceof different concentrations of a test compound. The ability of the testcompound to diminish binding of the compstatin analog to C3 isevaluated. A test compound that significantly diminishes binding of thecompstatin analog to C3 is a candidate compstatin mimetic. For example,a test compound that diminishes steady-state concentration of acompstatin analog-C3 complex, or that diminishes the rate of formationof a compstatin analog-C3 complex by at least 25%, or by at least 50%,is a candidate compstatin mimetic. One of skill in the art willrecognize that a number of variations of this screening assay may beemployed. Compounds to be screened include natural products, librariesof aptamers, phage display libraries, compound libraries synthesizedusing combinatorial chemistry, etc. The invention encompassessynthesizing a combinatorial library of compounds based upon the coresequence described above and screening the library to identifycompstatin mimetics. Any of these methods could also be used to identifynew compstatin analogs having higher inhibitory activity than compstatinanalogs tested thus far. It will be appreciated that compstatin mimeticscould be used in the cell-reactive compounds of the invention, and theinvention provides such cell-reactive compstatin mimetics.

VI. Cell-Reactive or Long-Acting Compstatin Analogs

The invention provides a variety of cell-reactive compstatin analogs. Insome aspects, a cell-reactive compstatin analog comprises a compound offormula A-L-M, wherein A is a moiety that comprises a cell-reactivefunctional group J, L is an optionally present linking portion, and Mcomprises a compstatin analog moiety. The compstatin analog moiety cancomprise any compstatin analog, e.g., any compstatin analog describedabove, in various embodiments. Formula A-L-M encompasses embodiments inwhich A-L is present at the N-terminus of the compstatin analog moiety,embodiments in which A-L is present at the C-terminus of the compstatinanalog moiety, embodiments in which A-L is attached to a side chain ofan amino acid of the compstatin analog moiety, and embodiments where thesame or different A-Ls are present at both ends of M. It will beappreciated that when certain compstatin analog(s) are present as acompstatin analog moiety in a compound of formula A-L-M, a functionalgroup of the compstatin analog will have reacted with a functional groupof L to form a covalent bond to A or L. For example, a cell-reactivecompstatin analog in which the compstatin analog moiety comprises acompstatin analog that contains an amino acid with a side chaincontaining a primary amine (NH₂) group (which compstatin analog can berepresented by formula R¹—(NH₂)), can have a formula R¹—NH-L-A in whicha new covalent bond to L (e.g., N—C) has been formed and a hydrogenlost. Thus the term “compstatin analog moiety” includes molecularstructures in which at least one atom of a compstatin analogparticipates in a covalent bond with a second moiety, which may, e.g.,modification of a side chain. Similar considerations apply to compstatinanalog moieties present in multivalent compound described above. In someembodiments, a blocking moiety at the N-terminus or C-terminus of acompstatin analog, e.g., a compstatin analog described in Section IVabove, is replaced by A-L in the structure of a cell-reactive compstatinanalog. In some embodiments, A or L comprises a blocking moiety. In someembodiments, a cell-reactive compstatin analog has a molar activity ofat least about 10%, 20%, or 30%, e.g., between 30% and 40%, between 30%and 50%, between 30% and 60%, between 30% and 70%, between 30% and 80%,between 30% and 90%, or more, of the activity of a correspondingcompstatin analog having the same amino acid sequence (and, ifapplicable, one or more blocking moiet(ies)) but not comprising acell-reactive moiety. In some embodiments in which a cell-reactivecompstatin analog comprises multiple compstatin analog moieties, themolar activity of the cell-reactive compstatin analog is at least about10%, 20%, or 30%, e.g., between 30% and 40%, between 30% and 50%,between 30% and 60%, between 30% and 70%, between 30% and 80%, between30% and 90%, or more, of the sum of the activities of said compstatinanalog moieties.

Cell-reactive moiety A can comprise any of a variety of differentcell-reactive functional groups J, in various embodiments. In general, acell-reactive functional group may be selected based at least in part onfactors such as (a) the particular functional group to be targeted; (b)the ability of the reactive functional group to react with the targetfunctional group under physiologically acceptable ex vivo conditions(e.g., physiologically acceptable pH and osmolarity) and/or in vivoconditions (e.g., in blood); (c) the specificity of the reaction betweenthe reactive functional group and the target functional group underphysiologically acceptable ex vivo conditions and/or in vivo; (d) thestability (e.g., under in vivo conditions) of the covalent bond thatwould result from reaction of the reactive functional group with itstarget functional group; (e) the ease of synthesizing a cell-reactivecompstatin analog comprising the reactive functional group, etc. In someembodiments, a reactive functional group that reacts with its targetchemical group without releasing a leaving group is selected. In someembodiments, a reactive functional group that results in release of aleaving group upon reaction with a target is selected. Compoundscontaining such groups may be useful, e.g., to monitor progress and/orextent of a reaction. In some embodiments, a leaving group isphysiologically acceptable to cells, tissues, or organs in the amountgenerated (e.g., based on concentration and/or absolute amountgenerated) and/or is medically acceptable to a subject in the amountgenerated in vivo (e.g., based on concentration in a relevant body fluidsuch as blood and/or based on the absolute amount generated). In someembodiments, a leaving group generated ex vivo is at least in partremoved, e.g., by washing cells or by washing or perfusing a tissue ororgan, e.g., with saline.

In many embodiments, a cell-reactive functional group of use in theinvention reacts with a side chain of an amino acid residue and/or withan N-terminal amino group or C-terminal carboxyl group of a protein. Insome embodiments, the cell-reactive functional group is reactive withsulfhydryl (—SH) groups, which are found in the side chains of cysteineresidues. In some embodiments, a maleimide group is used. Maleimidegroups react with sulfhydryl groups of cysteine residues of proteins atphysiologic pH and form a stable thioether linkage. In some embodiments,a haloacetyl group, such as an iodoacetyl or a bromoacetyl group, isused. Haloacetyls react with sulfhydryl groups at physiologic pH. Thereaction of the iodoacetyl group proceeds by nucleophilic substitutionof iodine with a sulfur atom from a sulfhydryl group resulting in astable thioether linkage. In other embodiments, an iodoacetamide groupis used. In some embodiments, the cell-reactive functional group reactswith amino (—NH₂) groups, which are present at the N-termini of proteinsand in the side chain of lysine residues (ε-amino group). In someembodiments an activated ester, e.g., a succinimidyl ester (i.e., NHSester) is used. For example, N-hydroxysuccinimide (NHS) or itswater-soluble analog (sulfo-NHS) can be used in the synthesis, wherebythe resulting cell-reactive compstatin analog comprises an NHS ester. Insome embodiments, the cell-reactive functional group reacts withcarboxyl (—COOH) groups, which are present at the C-termini of proteinsand in the side chains of various amino acid residues. In someembodiments, the cell-reactive compstatin analog is reactive withhydroxyl (—OH) groups, which are present in the side chains of variousamino acids and in carbohydrate moieties of glycosylated proteins.

In general, linking portion L can comprise any one or more aliphaticand/or aromatic moieties consistent with the formation of a stablecompound joining the linked moieties. The term “stable”, as used herein,preferably refers to compounds which possess stability sufficient toallow manufacture and which maintain the integrity of the compound for asufficient period of time, e.g., to be useful for one or more purposesdescribed herein. In some embodiments, L comprises a saturated orunsaturated, substituted or unsubstituted, branched or unbranched,aliphatic chain having a length of between 1 and 30, between 1 and 20,between 1 and 10, between 1 and 6, or 5 or less carbon atoms, wherelength refers to the number of C atoms in the main (longest) chain. Insome embodiments, the aliphatic chain comprises one or more heteroatoms(O, N, S), which may be independently selected. In some embodiments, atleast 50% of the atoms in the main chain of L are carbon atoms. In someembodiments, L comprises a saturated alkyl moiety (CH₂)., wherein n isbetween 1 and 30.

In some embodiments, L comprises one or more heteroatoms and has alength of between 1 and 1000, between 1 and 800, between 1 and 600,between 1 and 400, between 1 and 300, between 1 and 200, between 1 and100, between 1 and 50, between 1 and 30, or between 1 and 10 totalcarbon atoms in a chain. In some embodiments, L comprises anoligo(ethylene glycol) moiety (—(O—CH₂—CH₂—)_(n)) wherein n is between 1and 500, between 1 and 400, between 1 and 300, between 1 and 200,between 1 and 100, between 10 and 200, between 200 and 300, between 100and 200, between 40 and 500, between 30 and 500, between 20 and 500,between 10 and 500, between 1 and 40, between 1 and 30, between 1 and20, or between 1 and 10.

In some embodiments, L comprises an unsaturated moiety such as —CH═CH—or —CH₂—CH═CH—; a moiety comprising a non-aromatic cyclic ring system(e.g., a cyclohexyl moiety), an aromatic moiety (e.g., an aromaticcyclic ring system such as a phenyl moiety); an ether moiety (—C—O—C—);an amide moiety (—C(═O)—N—); an ester moiety (—CO—O—); a carbonyl moiety(—C(═O)—); an imine moiety (—C═N—); a thioether moiety (—C—S—C—); anamino acid residue; and/or any moiety that can be formed by the reactionof two compatible reactive functional groups. In certain embodiments,one or more moieties of a linking portion or cell-reactive moiety is/aresubstituted by independent replacement of one or more of the hydrogen(or other) atoms thereon with one or more moieties including, but notlimited to aliphatic; aromatic, aryl; alkyl, aralkyl, alkanoyl, aroyl,alkoxy; thio; F; Cl; Br; I; —NO2; —CN; —CF3; —CH2CF3; —CHCl2; —CH2OH;—CH2CH2OH; —CH2NH2; —CH2SO2CH3; — or —GRG1 wherein G is —O—, —S—,—NRG2—, —C(═O)—, —S(═O)—, —SO2—, —C(═O)O—, —C(═O)NRG2—, —OC(═O)—,—NRG2C(═O)—, —OC(═O)O—, —OC(═O)NRG2—, —NRG2C(═O)O—, —NRG2C(═O)NRG2—,—C(═S)—, —C(═S)S—, —SC(═S)—, —SC(═S)S—, —C(═NRG2)—, —C(═NRG2)O—,—C(═NRG2)NRG3—, —OC(═NRG2)—, —NRG2C(═NRG3)—, —NRG2SO2—, —NRG2SO2NRG3—,or —SO2NRG2—, wherein each occurrence of RG1, RG2 and RG3 independentlyincludes, but is not limited to, hydrogen, halogen, or an optionallysubstituted aliphatic, aromatic, or aryl moiety. It will be appreciatedthat cyclic ring systems when present as substituents may optionally beattached via a linear moiety. Combinations of substituents and variablesenvisioned by this invention are preferably those that result in theformation of stable compounds useful in any one or more of the methodsdescribed herein, e.g., useful for the treatment of one or moredisorders and/or for contacting a cell, tissue, or organ, as describedherein, and/or useful as intermediates in the manufacture of one or moresuch compounds.

L can comprise one or more of any of the moieties described in thepreceding paragraph, in various embodiments. In some embodiments, Lcomprises two or more different moieties linked to one another to form astructure typically having a length of between 1 to about 60 atoms,between 1 to about 50 atoms, e.g., between 1 and 40, between 1 and 30,between 1 and 20, between 1 and 10, or between 1 and 6 atoms, wherelength refers to the number of atoms in the main (longest) chain. Insome embodiments, L comprises two or more different moieties linked toone another to form a structure typically having between

-   -   to about 40, e.g., between 1 and 30, e.g., between 1 and 20,        between 1 and 10, or between 1 and 6 carbon atoms in the main        (longest) chain. In general, the structure of such a        cell-reactive compstatin analog can be represented by formula        A-(L^(Pj))j-M, wherein j is typically between 1 and 10, and each        L^(Pj) is independently selected from among the moieties        described in the preceding paragraph. In many embodiments, L        comprises one or more carbon-containing chains such as —(CH₂)n-        and/or —(O—CH₂—CH₂-)n, which are joined covalently to each other        and/or to a cell-reactive functional group or compstatin analog,        e.g., by moieties (e.g., amide, ester, or ether moieties) that        result from the reaction of two compatible reactive functional        groups. In some embodiments, L comprises an oligo(ethylene        glycol) moiety and/or a saturated alkyl chain. In some        embodiments, L comprises        —(CH₂)_(m)—C(═O)—NH—(CH₂CH₂O)_(n)(CH₂)_(p)C(═O)— or        —(CH₂)_(m)—C(═O)—NH—(CH₂)_(p)(OCH₂CH₂)_(n)C(═O)—. In some        embodiments, m, n, and p are selected so that the number of        carbons in the chain is between 1 and 500, e.g., between 2 and        400, between 2 and 300, between 2 and 200, between 2 and 100,        between 2 and 50, between 4 and 40, between 6 and 30, or between        8 and 20. In some embodiments, m is between 2 and 10, n is        between 1 and 500, and/or p is between 2 and 10. In some        embodiments, m is between 2 and 10, n is between 1 and 400,        and/or p is between 2 and 10. In some embodiments, m is between        2 and 10, n is between 1 and 300, and/or p is between 2 and 10.        In some embodiments, m is between 2 and 10, n is between 1 and        200, and/or p is between 2 and 10. In some embodiments, m is        between 2 and 10, n is between 1 and 100, and/or p is between 2        and 10. In some embodiments, m is between 2 and 10, n is between        1 and 50, and/or p is between 2 and 10. In some embodiments, m        is between 2 and 10, n is between 1 and 25, and/or p is between        2 and 10. In some embodiments, m is between 2 and 10, n is        between 1 and 8, and/or p is between 2 and 10. Optionally, at        least one —CH₂— is replaced by CH—R, wherein R can be any        substituent. Optionally, at least one —CH₂— is replaced by a        heteroatom, cyclic ring system, amide, ester, or ether moiety.        In some embodiments, L does not comprise an alkyl group having        more than 3 carbon atoms in the longest chain. In some        embodiments, L does not comprise an alkyl group having more than        4, 5, 6, 7, 8, 9, 10, or 11 carbon atoms in the longest chain.

In some embodiments of the invention, A comprises a cell-reactivefunctional group J and a linker L¹ comprising a linking portion L^(P1)and a reactive functional group that reacts with the compstatin analogto generate A-M In some embodiments, a bifunctional linker L² comprisingtwo reactive functional groups and a linking portion L^(P2) is used. Thereactive functional groups of L react with appropriate reactivefunctional groups of A and M to produce a cell-reactive compstatinanalog A-L-M. In some embodiments, the compstatin analog comprises alinker L³ comprising a linking portion L^(P3). For example, as discussedbelow, a linker comprising a reactive functional group may be present atthe N- or C-terminus or a moiety comprising a reactive functional groupmay be attached to the N- or C-terminus via a linker. Thus L may containmultiple linking portions L^(P) contributed, e.g., by A, by linker(s)used to join A and M, and/or by the compstatin analog. It will beunderstood that, when present in the structure A-L-M, certain reactivefunctional group(s) present prior to reaction in L¹, L², L³, etc., willhave undergone reaction, so that only a portion of said reactivefunctional group(s) will be present in the final structure A-L-M, andthe compound will contain moieties formed by reaction of said functionalgroups. In general, if a compound contains two or more linking portions,the linking portions can be the same or different, and can beindependently selected in various embodiments. Multiple linking portionsL^(P) can be attached to one another to form a larger linking portion L,and at least some of such linking portions can have one or morecompstatin analog(s) and/or cell-reactive functional group(s) attachedthereto. In molecules comprising multiple compstatin analogs, thecompstatin analogs can be the same or different and, if different, canbe independently selected. The same applies to the linking portions andreactive functional groups. The invention encompasses the use ofmultivalent compstatin analogs comprising one or more cell-reactivefunctional group(s) and use of concatamers of compstatin analogscomprising one or more cell-reactive functional group(s). In someembodiments, at least one linkage is a stable non-covalent linkage suchas a biotin/(strept)avidin linkage or other noncovalent linkage ofapproximately equivalent strength.

In some embodiments a cell-reactive compstatin analog comprises acompstatin analog in which any of SEQ ID NOs: 3-36 or 71-74 is extendedby one or more amino acids at the N-terminus, C-terminus, or both,wherein at least one of the amino acids has a side chain that comprisesa reactive functional group such as a primary or secondary amine, asulfhydryl group, a carboxyl group (which may be present as acarboxylate group), a guanidino group, a phenol group, an indole ring, athioether, or an imidazole ring. In some embodiments, the amino acid(s)is/are L-amino acids. In some embodiments, any one or more of the aminoacid(s) is a D-amino acid. If multiple amino acids are added, the aminoacids can be independently selected. In some embodiments, the reactivefunctional group (e.g., a primary or secondary amine) is used as atarget for addition of a moiety comprising a cell-reactive functionalgroup. Amino acids having a side chain that comprises a primary orsecondary amine include lysine (Lys) and diaminocarboxylic acids ofgeneral structure NH₂(CH₂)_(n)CH(NH₂)COOH such as 2,3-diaminopropionicacid (dapa), 2,4-diaminobutyric acid (daba), and ornithine (orn),wherein n=1 (dapa), 2 (daba), and 3 (orn), respectively. In someembodiments at least one amino acid is cysteine, aspartic acid, glutamicacid, arginine, tyrosine, tryptophan, methionine, or histidine. Cysteinehas a side chain comprising a sulfhydryl group. Aspartic acid andglutamic acid have a side chain comprising a carboxyl group (ionizableto a carboxylate group). Arginine has a side chain comprising aguanidino group. Tyrosine has a side chain comprising a phenol group(ionizable to a phenolate group). Tryptophan has a side chain comprisingan indole ring include, e.g., tryptophan. Methionine has a side chaincomprising a thioether group include, e.g., methionine. Histidine has aside chain comprising an imidazole ring. A wide variety of non-standardamino acids having side chains that comprise one or more such reactivefunctional group(s) are available, including naturally occurring aminoacids and amino acids not found in nature. See, e.g., Hughes, B. (ed.),Amino Acids, Peptides and Proteins in Organic Chemistry, Volumes 1-4,Wiley-VCH (2009-2011); Blaskovich, M., Handbook on Syntheses of AminoAcids General Routes to Amino Acids, Oxford University Press, 2010. Theinvention encompasses embodiments in which one or more non-standardamino acid(s) is/are used to provide a target for addition of a moietycomprising a cell-reactive functional group. Any one or more of theamino acid(s) may be protected as appropriate during synthesis of thecompound. For example, one or more amino acid(s) may be protected duringreaction(s) involving the target amino acid side chain. In someembodiments, wherein a sulfhydryl-containing amino acid is used as atarget for addition of a moiety comprising a cell-reactive functionalgroup, the sulfhydryl is protected while the compound is being cyclizedby formation of an intramolecular disulfide bond between other aminoacids such as cysteines.

In the discussion in this paragraph, an amino acid having a side chaincontaining an amine group is used as an example. The inventionencompasses analogous embodiments in which an amino acid having a sidechain containing a different reactive functional group is used. In someembodiments, an amino acid having a side chain comprising a primary orsecondary amine is attached directly to the N-terminus or C-terminus ofany of SEQ ID NOs: 3-36, 37, 71, 72, 73, or 74 or via a peptide bond. Insome embodiments, an amino acid having a side chain comprising a primaryor secondary amine is attached to the N- or C-terminus of any of SEQ IDNOs: 3-36, 37, 71, 72, 73, or 74 or via a linking portion, which maycontain any one or more of the linking moieties described above. In someembodiments, at least two amino acids are appended to either or bothtermini. The two or more appended amino acids may be joined to eachother by peptide bonds or at least some of the appended amino acids maybe joined to each other by a linking portion, which may contain any oneor more of the linking moieties described herein. Thus in someembodiments, a cell-reactive compstatin analog comprises a compstatinanalog moiety M of formula B1-R1-M₁-R2-B2, wherein M₁ represents any ofSEQ ID NOs: 3-36, 37, 71, 72, 73, or 74, either R1 or R2 may be absent,at least one of R1 and R2 comprises an amino acid having a side chainthat contains a primary or secondary amine, and B1 and B2 are optionallypresent blocking moieties. R1 and/or R2 may be joined to M₁ by a peptidebond or a non-peptide bond. R1 and/or R2 may comprise a linking portionL^(P3). For example, R1 can have formula M₂-L^(P3) and/or R2 can haveformula L^(P3)-M₂ wherein L^(P3) is a linking portion, and M₂ comprisesat least one amino acid having a side chain comprising a primary orsecondary amine. For example, M₂ can be Lys or an amino acid chaincomprising Lys. In some embodiments, L^(P3) comprises of consists of oneor more amino acids. For example, L^(P3) can be between 1 and about 20amino acids in length, e.g., between 4 and 20 amino acids in length. Insome embodiments, L^(P3) comprises or consist of multiple Gly, Ser,and/or Ala residues. In some embodiments, L^(P3) does not comprise anamino acid that comprises a reactive SH group, such as Cys. In someembodiments, L^(P3) comprises an oligo(ethylene glycol) moiety and/or asaturated alkyl chain. In some embodiments, L^(P3) is attached to theN-terminal amino acid of M₁ via an amide bond. In some embodiments,L^(P3) is attached to the C-terminal amino acid of M₁ via an amide bond.The compound may be further extended at either or both termini byaddition of further linking portion(s) and/or amino acid(s). The aminoacids can the same or different and, if different, can be independentlyselected. In some embodiments, two or more amino acids having sidechains comprising reactive functional groups are used, wherein thereactive functional groups can be the same or different. The two or morereactive functional groups can be used as targets for addition of two ormore moieties. In some embodiments, two or more cell-reactive moietiesare added. In some embodiments, a cell-reactive moiety and a targetingmoiety are added. In some embodiments, a linker and/or cell-reactivemoiety is attached to an amino acid side chain after incorporation ofthe amino acid into a peptide chain. In some embodiments, a linkerand/or cell-reactive moiety is already attached to the amino acid sidechain prior to use of the amino acid in the synthesis of a cell-reactivecompstatin analog. For example, a Lys derivative having a linkerattached to its side chain can be used. The linker may comprise acell-reactive functional group or may subsequently be modified tocomprise a cell-reactive functional group.

Certain cell-reactive compstatin analogs are described in further detailbelow In the following discussion, a peptide having the amino acidsequence

(SEQ ID NO: 37) Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr(corresponding to the compstatin analog of SEQ ID NO: 28, whereinasterisks in SEQ ID NO: 37 represent cysteines joined by a disulfidebond in the active compound, and (1Me)Trp represents1-methyl-tryptophan)), is used as an exemplary compstatin analog moiety;maleimide (abbreviated Mal) is used as an example of a cell-reactivefunctional group; (CH2)_(n) and (O-CH2-CH2)_(m) are used as examples oflinking portions; lysine is used as an example of an amino acidcomprising a reactive functional group (in some compounds), andacetylation and amidation of the N- and C-termini, respectively, areused as optionally present exemplary blocking moieties in some compoundsand are represented in italics, i.e., as Ac and NH₂ respectively. Itwill be appreciated that the compounds can be prepared using a varietyof synthetic approaches and using a variety of precursors. Thediscussion of various synthetic approaches and precursors below is notintended to limit the invention. In general, any of the features of anyof the compounds described below or herein can be freely combined withfeature(s) of other compounds described below or elsewhere herein, andthe invention encompasses such embodiments.

In some embodiments, the cell-reactive moiety is provided by acell-reactive compound comprising a maleimide group (as a cell-reactivefunctional group) and an alkanoic acid (RCOOH), where R is an alkylgroup. For example, 6-malemeidocaproic acid (Mal-(CH₂)₅—COOH), depictedbelow, can be used.

In some embodiments, the cell-reactive moiety is provided by aderivative of an alkanoic acid in which the carboxylic acid moiety hasbeen activated, e.g., the OH moiety has been converted to a betterleaving group. For example, the carboxyl group of compound I may bereacted with EDC, followed by reaction with NHS (which can optionally beprovided as water-soluble sulfo-NHS), resulting in anN-hydroxysuccinimide ester derivative of 6-malemeidocaproic acid, i.e.,6-maleimidohexanoic acid N-hydroxysuccinimide (NHS) ester (depictedbelow).

The compound of SEQ ID NO: 37 can be modified at the N- and/orC-terminus to generate a cell-reactive compstatin analog. For example,compound II can be used to generate the following cell-reactivecompstatin analog by reaction with the N-terminal amino group of Ile.

(SEQ ID NO: 38) Maleimide-(CH₂)₅—C(═O)-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-NH₂.It will be appreciated that in SEQ ID NO: 38 the —C(═O) moiety isattached to the immediately C-terminal amino acid (Ile), via a C—N bond,wherein the N is part of the amino acid and is not shown.

In other embodiments, a maleimide group is linked to Thr at theC-terminus, resulting in the following cell-reactive compstatin analog:

(SEQ ID NO: 39) Ac-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-(C═O)—(CH₂)₅-maleimide.

In some embodiments, a cell-reactive compstatin analog can besynthesized using bifunctional linker (e.g., a heterobifunctionallinker). An exemplary heterobifunctional linker comprising(CH₂—CH₂—O)_(n) and (CH₂)_(m) (where m=2) moieties is shown below:

Compound III comprises a maleimide group as a cell-reactive functionalgroup and an NHS ester moiety that reacts readily with an amino group(e.g., an N-terminal amino group or an amino group of an amino acid sidechain).

An embodiment of compound III in which n=2 can be used to generate thefollowing cell-reactive compstatin analog using the compstatin analog ofSEQ ID NO: 37:

(SEQ ID NO: 40) Maleimide-(CH₂)₂—C(═O)—NH—CH₂CH₂OCH₂CH₂OCH₂CH₂C(═O)-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg- Cys*-Thr-NH₂

It will be appreciated that in the compound of SEQ ID NO: 40 a —C(═O)moiety is attached to the N-terminal amino acid (Ile residue via a C—Nbond, wherein the N is part of the amino acid and is not shown. In someembodiments a linker has the formula of Compound III wherein n≥1.Exemplary values for n in a (CH₂—CH₂—O)_(n) moiety are provided herein.

In some embodiments, the alkyl chain that links the maleimide moiety tothe rest of the molecule contains more or fewer methylene units, theoligo(ethylene glycol) moiety contains more or fewer ethylene glycolunits, and/or there are more or fewer methylene units flanking either orboth sides of the oligo(ethylene glycol) moiety, as compared with thecompound of SEQ ID NO: 39 or SEQ ID NO: 40. Exemplary cell-reactivecompstatin analogs illustrative of a few such variations are presentedbelow (SEQ ID NOs: 41-46):

(SEQ ID NO: 41) Maleimide-(CH₂)₂—C(═O)—NH—CH₂CH₂OCH₂CH₂C(═O)-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg- Cys*-Thr-NH₂(SEQ ID NO: 42) Maleimide-(CH₂)₃—C(═O)—NH—CH₂CH₂OCH₂CH₂OCH₂C(═O)-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg- Cys*-Thr-NH₂(SEQ ID NO: 43) Maleimide-(CH₂)₅—C(═O)—NH—CH₂CH₂OCH₂CH₂OCH₂C(═O)-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg- Cys*-Thr-NH₂(SEQ ID NO: 44) Maleimide-(CH₂)₄—C(═O)—NH—CH₂CH₂OCH₂CH₂OCH₂CH₂C(═O)-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg- Cys*-Thr-NH₂(SEQ ID NO: 45) Maleimide-(CH₂)₂—C(═O)—NH—CH₂CH₂OCH₂CH₂OCH₂CH₂C(═O)-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg- Cys*-Thr-NH₂(SEQ ID NO: 46) Maleimide-(CH₂)₅—C(═O)—NH—CH₂CH₂OCH₂CH₂OCH₂C(═O)-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg- Cys*-Thr-NH₂

In some embodiments, SEQ ID NO: 37 is extended to comprise a Lys residueat the N- or C-terminus of the peptide, e.g., as exemplified below for aC-terminal linkage:

(SEQ ID NO: 47) Ac-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-Lys-NH₂.

In some embodiments, a Lys residue is attached to the N- or C-terminusof SEQ ID NO: 37 via a peptide linker, e.g., as exemplified below for aC-terminal linkage:

(SEQ ID NO: 48) Ac-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-(Gly)₅-Lys-NH₂.

In some embodiments, a linker comprising a primary or secondary amine isadded to the N- or C-terminus of a compstatin analog. In someembodiments, the linker comprises an alkyl chain and/or anoligo(ethylene glycol) moiety. For example, NH₂(CH₂CH₂O)nCH₂C(═O)OH(e.g., 8-amino-3,6-dioxaoctanoic acid (AEEAc) or11-amino-3,6,9-trioxaundecanoic acid) or an NHS ester thereof (e.g., anNHS ester of 8-amino-3,6-dioxaoctanoic acid or11-amino-3,6,9-trioxaundecanoic acid), can be used. In some embodiments,the resulting compound is as follows (wherein the portion contributed bythe linker is shown in bold):

(SEQ ID NO: 49) NH ₂ (CH ₂ ) ₅ C(═O)-Ile-Cys-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys-Thr-NH₂ (SEQ ID NO: 50) NH ₂ (CH ₂ CH ₂ O) ₂CH ₂ C(═O)-Ile-Cys-Val-(1Me)Trp- Gln-Asp-Trp-Gly-Ala-His-Arg-Cys-Thr-NH₂

In some embodiments, a Lys residue is attached to the N- or C-terminusof SEQ ID NO: 37 via a linker comprising a non-peptide portion. Forexample, the linker can comprise an alkyl chain, oligo(ethylene glycol)chain, and/or cyclic ring system. In some embodiments, 8-AEEAc or an NHSester thereof is used, resulting (in the case of attachment of Lys atthe C-terminus) in the following compound (wherein the portioncontributed by 8-AEEAc is shown in bold):

(SEQ ID NO: 51) Ac-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-NH — CH ₂ CH ₂ OCH ₂ CH ₂ OCH ₂ — C(═O)- Lys-NH₂

It will be appreciated that in SEQ ID NOs: 49 and 50, a —C(═O) moiety isattached to the adjacent Ile residue via a C—N bond, wherein the N ispart of the amino acid and is not shown. Similarly, in SEQ ID NO: 51, a—C(═O) moiety is attached to the adjacent Lys residue via a C—N bond,wherein the N is part of the amino acid and is not shown. It will alsobe appreciated that that in SEQ ID NO: 51 the NH moiety is attached tothe immediately N-terminal amino acid (Thr), via a C—N bond, wherein theC is the carbonyl carbon of the amino acid and is not shown.

The compounds of SEQ ID NOs: 47-51 can readily be modified at theprimary amine group to produce a cell-reactive compstatin analog. Forexample, the compounds of SEQ ID NOs: 47-51 (or other compoundscomprising a primary or secondary amine and a compstatin analog moiety)can be reacted with 6-maleimidocaproic acid N-succinimidyl ester toproduce the following cell-reactive compstatin analogs:

(SEQ ID NO: 52) Ac-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-Lys- (C(═O)—(CH₂)₅-Mal)-NH₂.(SEQ ID NO: 53) Ac-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-(Gly)₅-Lys-- (C(═O)—(CH₂)₅-Mal)-NH₂.(SEQ ID NO: 54) Mal-(CH₂)₅—(C(═O)—NH(CH2) ₅ C(═O)-Ile-Cys-Val-(1Me)Trp-Gln-Asp-Trp-Gly- Ala-His-Arg-Cys-Thr-NH₂(SEQ ID NO: 55) Mal-(CH₂)₅—(C(═O)NH(CH ₂ CH ₂ O) ₂ CH ₂ C(═O)-Ile-Cys-Val-(1Me)Trp-Gln-Asp- Trp-Gly-Ala-His-Arg-Cys-Thr-NH₂(SEQ ID NO: 56) Ac-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-NH — CH ₂ CH ₂ OCH ₂ CH ₂ OCH ₂ — C(═O)-Lys-(C(═O)—(CH₂)₅-Mal)-NH₂

In another embodiment, a cell-reactive compstatin analog is representedas:

(SEQ ID NO: 57) Ac- Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-Lys- C(═O)—CH₂(OCH₂CH2)₂NH(C(═O)—(CH₂)₅-Mal)-NH₂.

The invention provides variants of SEQ ID NOs: 38-57 in which

(SEQ ID NO: 75) -Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-is replaced by an amino acid sequence comprising the amino acid sequenceof any other compstatin analog, e.g., of any of SEQ ID NOs 3-27 or29-36, 37, 71, 72, 73, or 74 with the proviso that blocking moiet(ies)present at the N- and/or C-termini of a compstatin analog may be absent,replaced by a linker (which may comprise a blocking moiety), or attachedto a different N- or C-terminal amino acid present in the correspondingvariant(s).

Other bifunctional cross-linkers comprising a maleimide as acell-reactive moiety and an NHS ester as an amine-reactive moiety of usein various embodiments of the invention include, e.g., succinimidyl4-(p-maleimidophenyl)butyrate (SMPB); succinimidyl4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC);N-γ-maleimidobutyryl-oxysuccinimide ester (GMBS). Addition of asulfonate to the NHS ring results in water-soluble analogs such assulfo-succinimidyl(4-iodoacetyl)-aminobenzoate (sulfo-SIAB),sulfo-succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate(sulfo-SMCC), sulfo-succinimidyl 4-(p-maleimidophenyl)butyrate(sulfo-SMPB), sulfo-N-γ-maleimidobutyryl-oxysuccinimide ester(sulfo-GMBS) etc., which can avoid the need for an organic solvent. Insome embodiments, a long chain version of any of the foregoing,comprising a spacer arm between the NHS ester moiety and the remainderof the molecule, is used. The spacer can comprise, e.g., an alkyl chain.An example issuccinimidyl-4-[N-Maleimidomethyl]cyclohexane-1-carboxy-[6-amidocaproate].

In some embodiments, a bifunctional linker comprising an NHS ester (asan amine-reactive moiety) and an iodoacetyl group (reactive withsulfhydryl groups) is used. Such linkers include, e.g.,N-succinimidyl(4-iodoacetyl)-aminobenzoate (SIAB); succinimidyl6-[(iodoacetyl)-amino]hexanoate (SIAX); succinimidyl6-[6-(((iodoacetyl)amino)-hexanoyl) amino]hexanoate (SIAXX);succinimidyl 4-((iodoacetyl)amino)methyl)-cyclohexane-1-carboxylate(SIAC); succinimidyl(iodoacetyl)amino)methyl-cyclohexane-1-carbonyl)amino)hexanoate (SIACX);

In some embodiments, a bifunctional linker comprising an NHS ester (asan amine-reactive moiety) and a pyridy disulfide group (as acell-reactive moiety reactive with sulfhydryl groups) is used. Examplesinclude N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP);succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (SMPT) andversions comprising a sulfonate on the NHS ring and/or a spacercomprising an alkyl chain between the NHS ester moiety and the rest ofthe molecule (e.g., succinimidyl6-(3-[2-pyridyldithio]-propionamido)hexanoate) (LC-SPDP). Variations ofsuch linkers that include additional or different moieties could beused. For example, a longer or shorter alkyl chain could be used in aspacer, or an oligo(ethylene glycol) moiety instead of an alkyl chain.

In general, a cell-reactive compstatin analog can be synthesized using avariety of approaches. Cell-reactive compounds that comprise acell-reactive functional group and a linker can often be purchased aspreformed building blocks. For example, 6-malemeidocaproic acid and6-maleimidocaproic acid N-hydroxysuccinimide ester can be purchased fromvarious suppliers. Alternately, such compounds can be synthesized usingmethods known in the art. See, e.g., Keller 0, Rudinger J. Helv ChimActa. 58(2):531-41, 1975 and Hashida S, et al., J Appl Biochem.,6(1-2):56-63, 1984. See also, Hermanson, G. supra, and referencestherein, for discussion of methods and reagents of use for synthesizingconjugates. In general, the invention encompasses any method ofproducing a compound comprising a compstatin analog moiety and acell-reactive functional group, and the resulting compounds.

In some embodiments, an amino acid having a linker attached to a sidechain is used in the synthesis of a linear peptide. The linear peptidecan be synthesized using standard methods for peptide synthesis known inthe art, e.g., standard solid-phase peptide synthesis. The linearpeptide is then cyclized (e.g., by oxidation of the Cys residues to forman intramolecular disulfide). The cyclic compound may then be reactedwith a linker comprising a cell-reactive functional group. In otherembodiments, a moiety comprising a cell-reactive functional group isreacted with a linear compound prior to cyclization thereof. In general,reactive functional groups can be appropriately protected to avoidundesired reaction with each other during synthesis of a cell-reactivecompstatin analog. The cell-reactive functional group, any of the aminoacid side chains, and/or either or both termini of the peptide may beprotected during the reaction and subsequently deprotected. For example,SH groups of Cys residues and/or SH-reactive moieties such as maleimidescan be protected until after cyclization to avoid reaction between them.The reaction conditions are selected based at least in part on therequirements of the particular reactive functional group(s) to achievereasonable yield in a reasonable time period. Temperature, pH, and theconcentration of the reagents can be adjusted to achieve the desiredextent or rate of reaction. See, e.g., Hermanson, supra. The desiredproduct can be purified, e.g., to remove unreacted compound comprisingthe cell-reactive functional group, unreacted compstatin analog,linker(s), products other than the desired cell-reactive compstatinanalog that may have been generated in the reaction, other substancespresent in the reaction mixture, etc. Compositions and methods formaking the cell-reactive compstatin analogs, and intermediates in thesynthesis, are aspects of the invention.

In some aspects of the invention, linker(s) described above are used inthe production of compstatin analogs comprising a moiety such as apolyethylene glycol (PEG) chain or other polymer(s) that, e.g.,stabilize the compound, increase its lifetime in the body, increase itssolubility, decrease its immunogenicity, and/or increase its resistanceto degradation. Without limiting the invention in any way, such a moietymay be referred to herein as a “clearance reducing moiety” (CRM), and acompstatin analog comprising such a moiety may be referred to as a“long-acting compstatin analog”. In some embodiments, a long-actingcompstatin analog has an average plasma half-life of at least 1 day,e.g., 1-3 days, 3-7 days, 7-14 days, or 14-28 days, when administered IVat a dose of 10 mg/kg to humans or to non-human primates, or a dose ofabout 1-3 mg/kg, 3-5 mg/kg, 5-10 mg/kg, e.g., 7 mg/kg. In someembodiments, a long-acting compstatin analog has an average plasmahalf-life of at least 1 day, e.g., 1-3 days, 3-7 days, 7-14 days, or14-28 days, when administered subcutaneously at, e.g., a dose of about1-3 mg/kg, 3-5 mg/kg, 5-10 mg/kg, e.g., 7 mg/kg to humans or tonon-human primates. In some embodiments, a long-acting compstatin analoghas an average plasma half-life (e.g., a terminal half-life) of betweenabout 4-10, 5-9, 5-8, 6-9, 7-9, or 8-9 days, e.g., about 4, 4.5, 5, 5.5,6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 days when administeredintravenously at, e.g., a dose of about 1-3 mg/kg, 3-5 mg/kg, or 5-10mg/kg, e.g., 7 mg/kg to humans or to non-human primates. In someembodiments, a long-acting compstatin analog has an average plasmahalf-life (e.g., a terminal half-life) of between about 4-10, 5-9, 5-8,6-9, 7-9, or 8-9 days, e.g., about 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8,8.5, 9, 9.5 or 10 days, when administered subcutaneously at, e.g., adose of about 1-3 mg/kg, 3-5 mg/kg, 5-10 mg/kg, e.g., 7 mg/kg to humansor to non-human primates. In certain embodiments a long-actingcompstatin analog is characterized in that it is extensively absorbedfrom the site of administration during the time period followingsubcutaneous injection and provides, e.g., at or after about 1-2 daysfollowing administration, a blood level comparable to that which wouldbe achieved had the same amount of compound been administeredintravenously instead. In some embodiments, the blood level at or afterabout 2, 3, 4, 5, 6, 7, 8, or more days following administration of asubcutaneous dose is within about 5%, 10%, 15%, 20%, or 25% of the bloodlevel which would be achieved had the same amount of compound beenadministered intravenously instead. See, e.g., FIG. 11, showingpharmacokinetics of an intravenously and subcutaneously administereddose of an exemplary compound described herein after about 1-2 daysfollowing administration. In some embodiments, average plasma half-lifeof a long-acting compstatin analog following administration IV at a doseof 10 mg/kg to humans or to non-human primates is increased by at leasta factor of 2, e.g., by a factor of 2-5, 5-10, 10-50, or 50-100-fold or100-150-fold or 150-200 fold as compared with that of a correspondingcompstatin analog having the same amino acid sequence (and, ifapplicable, one or more blocking moiet(ies)) but not comprising the CRM.It will be understood that in various embodiments such an increase inhalf-life may be observed following administration via other routes suchas subcutaneous administration and/or using other doses, e.g., otherdoses described herein, e.g., 20 mg/kg. As noted above, in someembodiments a compstatin analog of any of SEQ ID NOs: 3-36, 37, 71, 72,73, or 74 is extended by one or more amino acids at the N-terminus,C-terminus, or both, wherein at least one of the amino acids has a sidechain that comprises a reactive functional group such as a primary orsecondary amine, a sulfhydryl group, a carboxyl group (which may bepresent as a carboxylate group), a guanidino group, a phenol group, anindole ring, a thioether, or an imidazole ring, which facilitateconjugation with a reactive functional group to attach a CRM to thecompstatin analog. It will be understood that a corresponding compstatinanalog not comprising the CRM may also lack one or more such amino acidswhich are present in the long-acting compstatin analog to which itcorresponds. Thus, a corresponding compstatin analog comprising any ofSEQ ID NOs: 3-36, 37, 71, 72, 73, or 74 and lacking a CRM will beunderstood to “have the same amino acid sequence” as SEQ ID NO: 3-36,37, 71, 72, 73, or 74, respectively. For example, a correspondingcompstatin analog comprising the amino acid sequence of SEQ ID NO: 14,21, 28, 29, 32, 33, 34, or 36 and lacking a CRM will be understood to“have the same amino acid sequence” as SEQ ID NO: 14, 21, 28, 29, 32,33, 34, or 36, respectively. In some embodiments, a plasma half-life isa terminal half-life after administration of a single IV dose. In someembodiments, a plasma half-life is a terminal half-life after steadystate has been reached following administration of multiple IV doses. Insome embodiments, a long-acting compstatin analog achieves a Cmax inplasma at least 5-fold as great as that of a corresponding compstatinanalog not comprising the CRM, e.g., between 5- and 50-fold as great,following administration of a single IV dose to a primate, or followingadministration of multiple IV doses. In some embodiments, a long-actingcompstatin analog achieves a Cmax in plasma between 10- and 20-fold asgreat as that of a corresponding compstatin analog not comprising theCRM following administration of a single IV dose to a primate, orfollowing administration of multiple IV doses. In some embodiments aprimate is human. In some embodiments a primate is a non-human primate,e.g., a monkey, such as a Cynomolgus monkey or Rhesus monkey. In someembodiments, renal clearance of a long-acting compstatin analog duringthe first 24 hours following administration IV at a dose of 10 mg/kg or20 mg/kg to humans or to non-human primates is reduced by at least afactor of 2, e.g., by a factor of 2-5, 5-10, 10-50, or 50-100-fold or100-150-fold or 150-200 fold as compared with renal clearance of acorresponding compstatin analog. It will be understood that in variousembodiments such a reduction in renal clearance may be observedfollowing administration via other routes such as subcutaneousadministration and/or using other doses, e.g., other doses describedherein, e.g., 20 mg/kg. The concentration of compstatin analog can bemeasured in blood and/or urine samples using, e.g., UV, HPLC, massspectrometry (MS) or antibody to the CRM, or combinations of suchmethods, such as LC/MS or LC/MS/MS. Pharmacokinetic parameters such ashalf-life and clearance can be determined using methods known to thoseof ordinary skill in the art. Pharmacokinetic analysis can be performed,e.g., with WinNonlin software v 5.2 (Pharsight Corporation, St. Louis,Mo.) or other suitable programs.

In certain embodiments a CRM is stable in physiological conditions forat least 24 hours or more. In certain embodiments a CRM is stable inmammalian, e.g., primate, e.g., human or non-human primate (e.g.,monkey) blood, plasma, or serum for at least 24 hours. In variousembodiments at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%,or more, of the CRM molecules remains intact upon incubation inphysiological conditions for 24 hours, 48 hours, 72 hours, 96 hours, 120hours, 144 hours, 168 hours, or more. In various embodiments at least50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more, of the CRMmolecules remains intact upon incubation in blood, plasma, or serum at37 degrees C. for 48 hours, 72 hours, 96 hours, 120 hours, 144 hours,168 hours, or more. Incubation may be performed using a CRM at aconcentration of between 1 microgram/ml to about 100 mg/ml in variousembodiments. Samples may be analyzed at various time points. Size orintactness may be assessed using, e.g., chromatography (e.g., HPLC),mass spectrometry, Western blot, or any other suitable method. Suchstability characteristics may be conferred on a moiety conjugated to theCRM. In various embodiments, a long-acting compstatin analog comprisinga CRM may have any of the afore-mentioned stability characteristics. Insome aspects intact with regard to a long-acting compstatin analog meansthat the compstatin analog moiety remains conjugated to the CRM and theCRM size remains about the same as at the start of incubation oradministration.

In some embodiments, a long-acting compstatin analog has a molaractivity of at least about 10%, 20%, 30%, e.g., between 30% and 40%,between 30% and 50%, between 30% and 60%, between 30% and 70%, between30% and 80%, between 30% and 90%, or more, of the activity of acorresponding compstatin analog having the same amino acid sequence(and, if applicable, one or more blocking moiet(ies)) but not comprisinga CRM. In some embodiments wherein a long-acting compstatin analogcomprises multiple compstatin analog moieties, the molar activity of thelong-acting compstatin analog is at least about 10%, 20%, or 30%, e.g.,between 30% and 40%, between 30% and 50%, between 30% and 60%, between30% and 70%, between 30% and 80%, between 30% and 90%, or more, of thesum of the activities of said compstatin analog moieties. In someembodiments, a polyethylene glycol (PEG) comprises a (CH₂CH₂O)_(n)moiety having a molecular weight of at least 500 daltons. In someembodiments, a linker described above comprises an (CH₂CH₂O)_(n) moietyhaving an average molecular weight of between about 500; 1,000; 1,500;2,000; 5,000; 10,000; 20,000; 30,000; 40,000; 50,000; 60,000; 70,000;80,000; 90,000; and 100,000 daltons. In some embodiments the averagemolecular weight of a PEG is at least 20,000 daltons, up to about100,000; 120,000; 140,000; 160,000; 180,000; or 200,000 daltons.“Average molecular weight” refers to the number average molecularweight. In some embodiments, the polydispersity D of a (CH₂CH₂O)n moietyis between 1.0005 and 1.50, e.g., between 1.005 and 1.10, 1.15, 1.20,1.25, 1.30, 1.40, or 1.50, or any value between 1.0005 and 1.50.

In some embodiments, a (CH₂CH₂O)n moiety is monodisperse and thepolydispersity of a (CH₂CH₂O)n moiety is 1.0. Such monodisperse(CH₂CH₂O)n moieties are known in the art and are commercially availablefrom Quanta BioDesign (Powell, Ohio), and include, by way of nonlimitingexample, monodisperse moieties where n is 2, 4, 6, 8, 12, 16, 20, or 24.

In some embodiments, a compound comprises multiple (CH₂CH₂O)_(n)moieties wherein the total molecular weight of said (CH₂CH₂O)_(n)moieties is between about 1,000; 5,000; 10,000; 20,000; 30,000; 40,000;50,000; 60,000; 70,000; 80,000; 90,000; and 100,000 daltons. In someembodiments the average total molecular weight of the compound or(CH₂CH₂O)_(n) moieties is at least 20,000 daltons, up to about 100,000;120,000; 140,000; 160,000; 180,000; or 200,000 daltons. In someembodiments, the compound comprises multiple (CH₂CH₂O)_(n) moietieshaving defined lengths, e.g., n=4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, or 30 or more. In some embodiments, the compound comprises asufficient number of (CH₂CH₂O)_(n) moieties having defined lengths toresult in a total molecular weight of said (CH₂CH₂O)_(n) moieties ofbetween about 1,000; 5,000; 10,000; 20,000; 30,000; 40,000; 50,000;60,000; 70,000; 80,000; 90,000; and 100,000 daltons. In some embodimentsthe average total molecular weight of the compound or (CH₂CH₂O)_(n)moieties is at least 20,000 daltons, up to about 100,000; 120,000;140,000; 160,000; 180,000; or 200,000 daltons. In some embodiments n isbetween about 30 and about 3000. In some embodiments a compstatin analogmoiety is attached at each end of a linear PEG. A bifunctional PEGhaving a reactive functional group at each end of the chain may be used,e.g., as described above. In some embodiments the reactive functionalgroups are identical while in some embodiments different reactivefunctional groups are present at each end. In some embodiments, multiple(CH₂CH₂O)_(n) moieties are provided as a branched structure. Thebranches may be attached to a linear polymer backbone (e.g., as acomb-shaped structure) or may emanate from one or more central coregroups, e.g., as a star structure. In some embodiments, a branchedmolecule has 3 to 10 (CH₂CH₂O)_(n) chains. In some embodiments, abranched molecule has 4 to 8 (CH₂CH₂O)_(n) chains. In some embodiments,a branched molecule has 10, 9, 8, 7, 6, 5, 4, or 3 (CH₂CH₂O)_(n) chains.In some embodiments, a star-shaped molecule has 10-100, 10-50, 10-30, or10-20 (CH₂CH₂O)_(n) chains emanating from a central core group. In someembodiments a long-acting compstatin analog thus may comprise, e.g.,3-10 compstatin analog moieties, e.g., 4-8 compstatin analog moieties,each attached to a (CH₂CH₂O)_(n) chain via a functional group at the endof the chain. In some embodiments a long-acting compstatin analog maycomprise, e.g., 10-100 compstatin analog moieties, each attached to a(CH₂CH₂O)_(n) chain via a functional group at the end of the chain. Insome embodiments, branches (sometimes referred to as “arms”) of abranched or star-shaped PEG contain about the same number of (CH₂CH₂O)moieties. In some embodiments, at least some of the branch lengths maydiffer. It will be understood that in some embodiments one or more(CH₂CH₂O)_(n) chains does not have a compstatin analog moiety attachedthereto. In some embodiments at least about 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, or 100% of the chains has a compstatin analog moietyattached thereto.

In genera and compounds depicted herein, a polyethylene glycol moiety isdrawn with the oxygen atom on the right side of the repeating unit orthe left side of the repeating unit. In cases where only one orientationis drawn, the present invention encompasses both orientations (i.e.,(CH₂CH₂O)_(n) and (OCH₂CH₂)_(n)) of polyethylene glycol moieties for agiven compound or genus, or in cases where a compound or genus containsmultiple polyethylene glycol moieties, all combinations of orientationsare encompasses by the present disclosure.

Formulas of some exemplary monofunctional PEGs comprising a reactivefunctional group are illustrated below. For illustrative purposes,formulas in which the reactive functional group(s) comprise an NHS esterare depicted, but other reactive functional groups could be used, e.g.,as described above. In some embodiments, the (CH₂CH₂O)_(n) are depictedas terminating at the left end with a methoxy group (OCH₃) but it willbe understood that the chains depicted below and elsewhere herein mayterminate with a different OR moiety (e.g., an aliphatic group, an alkylgroup, a lower alkyl group, or any other suitable PEG end group) or anOH group. It will also be appreciated that moieties other than thosedepicted may connect the (CH₂CH₂O)_(n) moieties with the NHS group invarious embodiments.

In some embodiments, a monofunctional PEG is of formula A:

wherein “Reactive functional group” and n are as defined above anddescribed in classes and subclasses herein;

-   R¹ is hydrogen, aliphatic, or any suitable end group; and-   T is a covalent bond or a C₁₋₁₂ straight or branched, hydrocarbon    chain wherein one or more carbon units of T are optionally and    independently replaced by —O—, —S—, —N(R^(x))—, —C(O)—, —C(O)O—,    —OC(O)—, —N(R^(x))C(O)—, —C(O)N(R^(x))—, —S(O)—, —S(O)₂—,    —N(R^(x))SO₂—, or —SO₂N(R^(x))—; and    each R^(x) is independently hydrogen or C₁₋₆ aliphatic.

Exemplary monofunctional PEGs of formula A include:

In Formula I, the moiety comprising the reactive functional group hasthe general structure —CO—(CH₂)_(m)—COO—NHS, where m=2. In someembodiments, a monofunctional PEGs has the structure of Formula I, wherem is between 1 and 10, e.g., between 1 and 5. For example, in someembodiments m is 3, as shown below:

In Formula II, the moiety comprising the reactive functional group hasthe general structure —(CH₂)_(m)—COO—NHS, where m=1. In some embodimentsa monofunctional PEG has the structure of Formula II, where m is between1 and 10 (e.g., wherein m is 5 as shown in Formula III below), orwherein m is 0 (as shown below in Formula IIIa).

In some embodiments a bifunctional linear PEG comprises a moietycomprising a reactive functional group at each of its ends. The reactivefunctional groups may be the same (homobifunctional) or different(heterobifunctional). In some embodiments the structure of abifunctional PEG may be symmetric, wherein the same moiety is used toconnect the reactive functional group to oxygen atoms at each end of the—(CH₂CH₂O)_(n) chain. In some embodiments different moieties are used toconnect the two reactive functional groups to the PEG portion of themolecule. The structures of exemplary bifunctional PEGs are depictedbelow. For illustrative purposes, formulas in which the reactivefunctional group(s) comprise an NHS ester are depicted, but otherreactive functional groups could be used.

In some embodiments, a bifunctional linear PEG is of formula B:

wherein each T and “Reactive functional group” is independently asdefined above and described in classes and subclasses herein, and n isas defined above and described in classes and subclasses herein.

Exemplary bifunctional PEGs of formula B include:

In Formula IV, the moiety comprising the reactive functional group hasthe general structure —(CH₂)_(m)—COO—NHS, where m=1. In someembodiments, a bifunctional PEG has the structure of Formula IV, where mis between 1 and 10, e.g., between 1 and 5. In certain embodiments m is0, e.g., embodiments the moiety comprising the reactive functional grouphas the general structure —COO—NHS. For example, in some embodiments abifunctional PEG has the structure of Formula IVa, as shown below:

In Formula V, the moiety comprising the reactive functional group hasthe general structure —CO—(CH₂)_(m)—COO—NHS, where m=2. In someembodiments, a bifunctional PEGs has the structure of Formula V, where mis between 1 and 10, e.g., between 1 and 5. In certain embodiments, forexample, m is 2, as shown below:

In some embodiments, the present invention provides a compstatin analogconjugated to a polymer. In certain embodiments, the present inventionprovides compstatin analog conjugates of PEG-containing compounds andgenera depicted herein. In some embodiments, a functional group (forexample, an amine, hydroxyl, or thiol group) on a compstatin analog isreacted with a PEG-containing compound having a “reactive functionalgroup” as described herein, to generate such conjugates. By way ofexample, Formulae III and IV, respectively, can form compstatin analogconjugates having the structure:

wherein,

represents the attachment point of an amine group on a compstatinanalog. In certain embodiments, an amine group is a lysine side chaingroup.

It will be appreciated that corresponding conjugates can be formed withany of the PEG-containing compounds and genera depicted herein,depending on the choice of reactive functional group and/or compstatinfunctional group. For example, Formulae IVa and Va, respectively, canform compstatin analog conjugates having the following structures

In certain embodiments, the PEG component of such conjugates has anaverage molecular weight of between about 20 kD-100 kD, about 20 kD-90kD, about 20 kD-80 kD, about 20 kD-70 kD, about 20 kD-60 kD, about 20kD-50 kD, about 30 kD-80 kD, about 30 kD-70 kD, about 30 kD-60 kD, about30 kD-50 kD, about 30 kD-45 kD, about 35 kD-50 kD, about 35 kD-45 kD,about 36 kD-44 kD, about 37 kD-43 kD, about 38 kD-42 kD, or about 39kD-41 kD. In certain embodiments, the PEG component of such conjugateshas an average molecular weight of about 40 kD. The term “bifunctional”or “bifunctionalized” is sometimes used herein to refer to a compoundcomprising two compstatin analog moieties linked to a CRM. Suchcompounds may be designated with the letter “BF”. In some embodiments abifunctionalized compound is symmetrical. In some embodiments thelinkages between the CRM and each of the compstatin analog moieties of abifunctionalized compound are the same. In some embodiments, eachlinkage between a CRM and a compstatin analog of a bifunctionalizedcompound comprises a carbamate. In some embodiments, each linkagebetween a CRM and a compstatin analog of a bifunctionalized compoundcomprises a carbamate and does not comprise an ester. In someembodiments, each compstatin analog of a bifunctionalized compound isdirectly linked to a CRM via a carbamate. In some embodiments, eachcompstatin analog of a bifunctionalized compound is directly linked to aCRM via a carbamate, and the bifunctionalized compound has thestructure:

In some embodiments of formulae and embodiments described herein,

represents point of attachment of a lysine side chain group in acompstatin analog having the structure:

wherein the symbol “

” denotes the point of attachment of a chemical moiety to the remainderof a molecule or chemical formula.

In some embodiments, a branched, comb, or star-shaped PEG comprises amoiety comprising a reactive functional group at the end of each ofmultiple —(CH₂CH₂O)_(n) chains. The reactive functional groups may bethe same or there may be at least two different groups. In someembodiments, a branched, comb, or star-shaped PEG is of the followingformulae:

wherein each R² is independently a “Reactive functional group” or R¹,and each T, n, and “Reactive functional group” is independently asdefined above and described in classes and subclasses herein. Thestructure of exemplary branched PEGs (having 8 arms, or branches)comprising NHS moieties as reactive functional groups is depicted below:

The structure of exemplary branched PEGs (having 4 arms, or branches)comprising NHS moieties as reactive functional groups is depicted below:

The number of branches emanating from the backbone may be varied. Forexample, the number 4 in the above formulae VI and VII may be changed toany other integer between 0 and 10 in various embodiments. In certainembodiments, one or more branches does not contain a reactive functiongroup and the branch terminates with a —CH₂CH₂OH or —CH₂CH₂OR group, asdescribed above.

In some embodiments a branched PEG has the structure of Formula VII,VIII, or IX (or variants thereof having different numbers of branches)with the proviso that x is

In some embodiments a branched PEG has the structure of Formula VII,VIII, or IX (or variants thereof having different numbers of branches)with the proviso that x is

Of course the methylene (CH₂) group in the above x moiety may insteadcomprise a longer alkyl chain (CH₂)_(m), where m is up to 2, 3, 4, 5, 6,8, 10, 20, or 30, or may comprise one or more other moieties describedherein.

In some embodiments, exemplary branched PEGs having NHS or maleimidereactive groups are depicted below:

In some embodiments, a variant of Formula X or XI are used, wherein 3 oreach of the 4 branches comprise a reactive functional group.

Still other examples of PEGS may be represented as follows:

As noted above, it will be appreciated that, as described herein, invarious embodiments any of a variety of moieties may be incorporatedbetween the peptide component and (CH₂CH₂O)_(n)—R moiety of along-acting compstatin analog, such as an linear alkyl, ester, amide,aromatic ring (e.g., a substituted or unsubstituted phenyl), asubstituted or unsubstituted cycloalkyl structure, or combinationsthereof. In some embodiments such moiet(ies) may render the compoundmore susceptible to hydrolysis, which may release the peptide portion ofthe compound from the CRM. In some embodiments, such release may enhancethe in vivo tissue penetration and/or activity of the compound. In someembodiments hydrolysis is general (e.g., acid-base) hydrolysis. In someembodiments hydrolysis is enzyme-catalyzed, e.g., esterase-catalyzed. Ofcourse both types of hydrolysis may occur. Examples of PEGs comprisingone or more such moieties and an NHS ester as a reactive functionalgroup are as follows:

In some embodiments a branched (multi-arm) PEG or star-shaped PEGcomprises a pentaerythritol core, hexaglycerin core, ortripentaerythritol core. It will be understood that the branches may notall emanate from a single point in certain embodiments.

Monofunctional, bifunctional, branched, and other PEGs comprising one ormore reactive functional groups may, in some embodiments, be obtainedfrom, e.g., NOF America Corp. White Plains, N.Y. or BOC Sciences 45-16Ramsey Road Shirley, N.Y. 11967, USA, among others, or may be preparedusing methods known in the art.

In some embodiments, a linkage between a CRM and a compstatin analogcomprises a carbamate. In some embodiments, a compstatin analog isdirectly linked to a CRM via a carbamate. In some embodiments, a linkagebetween a CRM and a compstatin analog does not comprise an ester. Insome embodiments, a linkage between a CRM and a compstatin analogcomprises a carbamate and does not comprise an ester. In someembodiments, a linkage between a CRM and a compstatin analog comprises acarbamate and does not comprise a bond that is more susceptible tohydrolysis in aqueous medium than a carbamate. In some embodiments theCRM comprises or consists of a PEG moiety.

In some embodiments, a linkage between a CRM and a compstatin analogcomprises an amide. In some embodiments, a compstatin analog is directlylinked to a CRM via an amide. In some embodiments, a linkage between aCRM and a compstatin analog comprises an amide and does not comprise anester. In some embodiments, a linkage between a CRM and a compstatinanalog comprises an amide and does not comprise a bond that is moresusceptible to hydrolysis in aqueous medium than an amide. In someembodiments the CRM comprises or consists of a PEG moiety.

In some embodiments, one or more compstatin analogs of amultifunctionalized compound (e.g., a bifunctionalized,trifunctionalized, or more extensively functionalized compound) islinked to a CRM by a linkage comprising a carbamate. In someembodiments, one or more compstatin analogs of a multifunctionalizedcompound (e.g., a bifunctionalized, trifunctionalized, or moreextensively functionalized compound) is linked to a CRM by a linkagethat does not comprise an ester. In some embodiments, one or morecompstatin analogs of a multifunctionalized compound (e.g., abifunctionalized, trifunctionalized, or more extensively functionalizedcompound) is linked to a CRM by a linkage that comprises a carbamate anddoes not comprise an ester. In some embodiments, one or more compstatinanalogs of a multifunctionalized compound (e.g., a bifunctionalized,trifunctionalized, or more extensively functionalized compound) islinked to a CRM by a linkage that comprises a carbamate and does notcomprise a bond that is more susceptible to hydrolysis in aqueous mediumthan a carbamate. In some embodiments, each compstatin analog of amultifunctionalized compound (e.g., a bifunctionalized,trifunctionalized, or more extensively functionalized compound) isdirectly linked to a CRM via a carbamate.

In some embodiments the CRM comprises or consists of a PEG moiety. Insome embodiments, one or more compstatin analogs of amultifunctionalized compound (e.g., a bifunctionalized,trifunctionalized, or more extensively functionalized compound) islinked to a CRM by a linkage comprising an amide. In some embodiments,one or more compstatin analogs of a multifunctionalized compound (e.g.,a bifunctionalized, trifunctionalized, or more extensivelyfunctionalized compound) is linked to a CRM by a linkage that comprisesan amide and does not comprise an ester. In some embodiments, one ormore compstatin analogs of a multifunctionalized compound (e.g., abifunctionalized, trifunctionalized, or more extensively functionalizedcompound) is linked to a CRM by a linkage that comprises an amide anddoes not comprise a bond that is more susceptible to hydrolysis inaqueous medium than an amide. In some embodiments, each compstatinanalog of a multifunctionalized compound (e.g., a bifunctionalized,trifunctionalized, or more extensively functionalized compound) isdirectly linked to a CRM via an amide. In some embodiments the CRMcomprises or consists of a PEG moiety.

In some embodiments, the present invention provides a compstatin analogconjugated with a polymer, wherein the polymer is other than PEG. Insome embodiments, a polymer is a polyoxazoline (POZ). Exemplary mono-and poly-functionalized polyoxazoline derivatives for directconjugation, or for conjugation via a linker, are depicted below:

Z—T—[N(COR^(x))CH₂CH₂]_(n)—T—R¹;

R¹—{[N(CO—T—Z)CH₂CH₂]_(m)[N(COR^(x))CH₂CH₂]_(n)}^(a)—T—R¹;

R¹—{[N(CO—T—Z¹)CH₂CH₂]_(p)—[N(COR^(x))CH₂CH₂]_(n)—[N(CO—T—Z²)CH₂CH₂]_(m)}^(a)—T—R¹;

R¹—{[N(CO—T—Z¹)CH₂CH₂]_(p)—[N(COR^(x))CH₂CH₂]_(n)—[N(CO—T—Z²)CH₂CH₂]_(m)}^(a)—T—Z;

R¹—[N(COR^(x))CH₂CH₂]_(n)—T—B(—R¹)(—T—Z)—T—[N(COR^(x))CH₂CH₂]_(m)—R¹;

wherein:

-   -   each of Z, Z¹ and Z² is independently a reactive functional        group as defined above and described in classes and subclasses        herein;    -   each of T, R^(x), and R¹ is independently as defined above and        described in classes and subclasses herein;    -   each of m, n, and p is independently an integer 0-1000, with the        limitation that the sum of m, n, and p for each formula is not        0;    -   a is “ran,” which indicates a random copolymer, or “block,”        which indicates a block copolymer;    -   B is a branching moiety that is linked with or without a linker        to the other parts of the polymer.        Other examples of functionalized polyoxazoline derivatives for        conjugation are extensively described in the art, including but        not limited to those described in PCT Patent Application        Publication Nos. WO/2010/006282, WO/2009/089542, WO/2009/043027        and WO/2008/106186, the entirety of each of which is hereby        incorporated by reference.

Exemplary compstatin analog conjugates with polyoxazoline polymers aredepicted below:

wherein each variable is independently as defined above and described inclasses and subclasses herein.

In some embodiments, the present invention provides a compstatin analogconjugated with a polymer, wherein the compstatin analog is connected tothe polymer via one or more linkers. In some embodiments, a polymer isselected from PEG-containing compounds and genera described above and inclasses and subclasses herein. In some embodiments, the presentinvention provides compstatin analog conjugates of PEG-containingcompounds and genera depicted herein, wherein the compstatin analog isconnected to the PEG-containing moieties via one or more linkers. Mono-and poly-functional PEGs that comprise one or more reactive functionalgroups for conjugation are defined above and described in classes andsubclasses herein, including but not limited to those of formula A, I,Ia, II, III, Ma, B, IV, IVa, V, Va, C, D, E, F, G, H, VI, VII, VIII, IX,X, XI, XII, XIII, XIV, XV, or XVI.

Suitable linkers for connecting a compstatin analog and a polymer moietysuch as PEG or polyoxazoline are extensively described above and inclasses and subclasses herein. In some embodiments, a linker hasmultiple functional groups, wherein one functional group is connected toa compstatin analog and another is connected to a polymer moiety. Insome embodiments, a linker is a bifunctional compound. In someembodiments, a linker has the structure of NH₂(CH₂CH₂O)nCH₂C(═O)OH,wherein n is 1 to 1000. In some embodiments, a linker is8-amino-3,6-dioxaoctanoic acid (AEEAc). In some embodiments, a linker isactivated for conjugation with a polymer moiety or a functional group ofa compstatin analog. For example, in some embodiments, the carboxylgroup of AEEAc is activated before conjugation with the amine group ofthe side chain of a lysine group.

In some embodiments, a suitable functional group (for example, an amine,hydroxyl, thiol, or carboxylic acid group) on a compstatin analog isused for conjugation with a polymer moiety, either directly or via alinker. In some embodiments, a compstatin analog is conjugated throughan amine group to a PEG moiety via a linker. In some embodiments, anamine group is the α-amino group of an amino acid residue. In someembodiments, an amine group is the amine group of the lysine side chain.In some embodiments, a compstatin analog is conjugated to a PEG moietythrough the amino group of a lysine side chain (6-amino group) via alinker having the structure of NH₂(CH₂CH₂O)nCH₂C(═O)OH, wherein n is 1to 1000. In some embodiments, a compstatin analog is conjugated to thePEG moiety through the amino group of a lysine side chain via an AEEAclinker. In some embodiments, the NH₂(CH₂CH₂O)nCH₂C(═O)OH linkerintroduces a —NH(CH₂CH₂O)nCH₂C(═O)— moiety on a compstatin lysine sidechain after conjugation. In some embodiments, the AEEAc linkerintroduces a —NH(CH₂CH₂O)₂CH₂C(═O)— moiety on a compstatin lysine sidechain after conjugation.

In some embodiments, a compstatin analog is conjugated to a polymermoiety via a linker, wherein the linker comprises an AEEAc moiety and anamino acid residue. In some embodiments, a compstatin analog isconjugated to a polymer moiety via a linker, wherein the linkercomprises an AEEAc moiety and a lysine residue. In some embodiments, apolymer is PEG. In some embodiments, the C-terminus of a compstatinanalog is connected to the amino group of AEEAc, and the C-terminus ofAEEAc is connected to a lysine residue. In some embodiments, theC-terminus of a compstatin analog is connected to the amino group ofAEEAc, and the C-terminus of AEEAc is connected to the α-amino group ofa lysine residue. In some embodiments, the C-terminus of a compstatinanalog is connected to the amino group of AEEAc, the C-terminus of AEEAcis connected to the α-amino group of the lysine residue, and a polymermoiety, such as a PEG moiety, is conjugated through the ε-amino group ofsaid lysine residue. In some embodiments, the C-terminus of the lysineresidue is modified. In some embodiments, the C-terminus of the lysineresidue is modified by amidation. In some embodiments, the N-terminus ofa compstatin analog is modified. In some embodiments, the N-terminus ofa compstatin analog is acetylated.

Exemplary conjugates comprising an AEEAc linker and a polymer aredepicted below, wherein

represents the attachment point of an amine group on a compstatinanalog,

represents a compstatin analog attaching through its C-terminus, andwherein each of the other variables is independently as defined aboveand described in classes and subclasses herewith. In some embodiments,an amine group is the amino group of a lysine side chain.

In certain embodiments a compstatin analog may be represented asM-AEEAc-Lys-B₂, wherein B₂ is a blocking moiety, e.g., NH₂, M representsany of SEQ ID NOs: 3-36, 37, 71, 72, 73, or 74, with the proviso thatthe C-terminal amino acid of any of SEQ ID NOs: 3-36, 37, 71, 72, 73, or74 is linked via a peptide bond to AEEAc-Lys-B₂. The NHS moiety of amonofunctional or multifunctional (e.g., bifunctional) PEG reacts withthe free amine of the lysine side chain to generate a monofunctionalized(one compstatin analog moiety) or multifunctionalized (multiplecompstatin analog moieties) long-acting compstatin analog. In variousembodiments any amino acid comprising a side chain that comprises areactive functional group may be used instead of Lys (or in addition toLys). A monofunctional or multifunctional PEG comprising a suitablereactive functional group may be reacted with such side chain in amanner analogous to the reaction of NHS-ester activated PEGs with Lys.

With regard to any of the above formulae and structures, it is to beunderstood that embodiments in which the compstatin analog componentcomprises any compstatin analog described herein, e.g., any compstatinanalog of SEQ ID NOs: 3-36, 37, 71, 72, 73, or 74, are expresslydisclosed. For example, and without limitation, a compstatin analog maycomprise the amino acid sequence of SEQ ID NO: 28. An exemplarylong-acting compstatin analog in which the compstatin analog componentcomprises the amino acid sequence of SEQ ID NO: 28 is depicted in FIG.10(C). It will be understood that the PEG moiety may have a variety ofdifferent molecular weights or average molecular weights in variousembodiments, as described herein. For example, individual PEG chainswithin a preparation may vary in molecular weight and/or differentpreparations may have different average molecular weights and/orpolydispersity, as described herein. In certain embodiments, the PEGmoiety in the compound of FIG. 10(C) has an average molecular weight ofbetween about 20 kD-100 kD, about 20 kD-90 kD, about 20 kD-80 kD, about20 kD-70 kD, about 20 kD-60 kD, about 20 kD-50 kD, about 30 kD-80 kD,about 30 kD-70 kD, about 30 kD-60 kD, about 30 kD-50 kD, about 30 kD-45kD, about 35 kD-50 kD, about 35 kD-45 kD, about 36 kD-44 kD, about 37kD-43 kD, about 38 kD-42 kD, or about 39 kD-41 kD. In some embodimentsthe PEG moiety in the compound of FIG. 10(C) has an average molecularweight between about 30 kD and about 50 kD, e.g., between about 35 kDand about 45 kD, between about 37.5 kD and about 42.5 kD. In certainembodiments in which the PEG moiety has an average molecular weight ofabout 40 kD, e.g., 37.5 kD-42.5 kD, 38 kD, 39 kD, 40 kD, 41 kD, 42 kD,the compound is sometimes referred to herein as CA28-2TS-BF. In certainembodiments a compound comprising a CRM, e.g., a PEG moiety, that has anaverage molecular weight of about 40 kD, e.g., 37.5 kD-42.5 kD, 38 kD,39 kD, 40 kD, 41 kD, 42 kD, the compound has a terminal half-life of atleast about 5 days, e.g., about 5-10 days, e.g., about 5, 6, 7, 8, 9days, when administered IV or subcutaneously to non-human primates orhumans, e.g., at a dose of about 1-3 mg/kg, 3-5 mg/kg, or 5-10 mg/kg.

In some aspects, the present invention relates to use of click chemistryin connection with compstatin analogs. “Click chemistry” is well knownin the art and is useful in some aspects of the present invention. Clickchemistry embodies, in certain embodiments, versatile cycloadditionreactions between azides and alkynes that enable a number of usefulapplications. Methods of carrying out click chemistry are known in theart, and are described by Kolb, H. C.; Sharpless, K. B., Drug Disc.Today, 2003, 1128-1137; Moses, J. E.; Moorhouse, A. D.; Chem. Soc. Rev.,2007, 1249-1262; the entire contents of each are hereby incorporated byreference. Click chemistry is a popular method of bioconjugation due toits high reactivity and selectivity, even in biological media. See Kolb,H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem. Int. Ed. 2001, 40,2004-2021; and Wang, Q.; Chan, T. R.; Hilgraf, R.; Fokin, V. V.;Sharpless, K. B.; Finn, M. G. J. Am. Chem. Soc. 2003, 125, 3192-3193. Inaddition, currently available recombinant techniques and syntheticmethods permit the introduction of azides and alkyne-bearingnon-canonical amino acids into peptides, proteins, cells, viruses,bacteria, and other biological entities that consist of or displayproteins. See Link, A. J.; Vink, M. K. S.; Tirrell, D. A. J. Am. Chem.Soc. 2004, 126, 10598-10602; Deiters, A.; Cropp, T. A.; Mukherji, M.;Chin, J. W.; Anderson, C.; Schultz, P. G. J. Am. Chem. Soc. 2003, 125,11782-11783.

As used herein, the term “click chemistry group” is sometimes used torefer\ to a reactive functional group capable of participating in aclick chemistry reaction with an appropriate second reactive functionalgroup, which second reactive functional group is also a click chemistrygroup. The first and second click chemistry groups, or entities (e.g.,molecules) comprising such groups, may be referred to as complementary.First and second entities, e.g., molecules, that comprise complementaryclick chemistry groups may be referred to as click chemistry partners.An entity or molecule comprising a click chemistry group may be referredto as “click-functionalized”. A bond formed by reaction of complementaryclick chemistry partners may be referred to as a “click chemistry bond”.

In some embodiments, the present invention provides click-functionalizedcompstatin analogs for, e.g., conjugation to a complementary moiety on apartner molecule or biomolecule. In some embodiments, a complementarypartner molecule or biomolecule is a polymer, peptide, protein, or amolecule that functions as a clearance-reducing moiety. In someembodiments, the “click-functionalized” moiety is an alkyne or an alkynederivative which is capable of undergoing [3+2] cycloaddition reactionswith complementary azide-bearing molecules and biomolecules. In anotherembodiment, the “click-functionalized” functionality is an azide or anazide derivative which is capable of undergoing [3+2] cycloadditionreactions with complementary alkyne-bearing molecules and biomolecules(i.e. click chemistry).

In some embodiments, a click-functionalized compstatin analog bears anazide group on any side chain group of the compstatin analog. In someembodiments, a click-functionalized compstatin analog bears an azidegroup on a lysine side chain group.

In some embodiments, a click-functionalized compstatin analog bears analkyne group on any side chain group of the compstatin compstatinanalog. In some embodiments, a click-functionalized compstatin analogbears an alkyne group on a lysine side chain group.

In some embodiments, the present invention provides compstatinconjugates comprising a compstatin analog, a molecule that functions asa clearance-reducing moiety, and a triazole linker. In some embodiments,a triazole linker is the result of click conjugation chemistry between acompstatin conjugate and a molecule that functions as aclearance-reducing moiety. In some embodiments the CRM may be any CRMdisclosed herein. For example, the CRM may be a PEG, a polypeptide, or aPOZ.

In some embodiments, the present invention provides compstatinconjugates comprising a compstatin analog, a PEG moiety, and a triazolelinker. In some embodiments, a triazole linker is the result of clickconjugation chemistry between a compstatin conjugate and a PEG moiety.

In some embodiments, the present invention provides compstatinconjugates comprising a compstatin analog, a polyoxazoline moiety, and atriazole linker. In some embodiments, a triazole linker is the result ofclick conjugation chemistry between a compstatin conjugate and apolyoxazoline moiety.

In some embodiments, click chemistry between a compstatin analog andanother moiety is transition metal catalyzed. Copper-containingmolecules which catalyze the “click” reaction include, but are notlimited to, copper wire, copper bromide (CuBr), copper chloride (CuCl),copper sulfate (CuSO₄), copper sulfate pentahydrate (CuSO₄.5H₂O), copperacetate (Cu₂(AcO₄), copper iodide (CuI), [Cu(MeCN)₄](OTf),[Cu(MeCN)₄](PF₆), colloidal copper sources, and immobilized coppersources. In some embodiments other metals, such as ruthenium. Reducingagents as well as organic and inorganic metal-binding ligands can beused in conjunction with metal catalysts and include, but are notlimited to, sodium ascorbate, tris(triazolyl)amine ligands,tris(carboxyethyl)phosphine (TCEP), sulfonated bathophenanthrolineligands, and benzimidazole-based ligands.

In some embodiments, compstatin analogs are conjugated to other moietiesusing metal free click chemistry (also known as copper free clickchemistry) to give a metal free composition or conjugates. In contrastto standard click chemistry, also known as copper assisted clickchemistry (CuACC), metal free click chemistry occurs between either astrained, cyclic alkyne or an alkyne precursor such as anoxanorbornadiene, and an azide group. As the name implies, no metalcatalyst is necessary for the reaction to occur. Examples of suchchemistries include reactions involving cyclooctyne derivatives(Codelli, et. al. J. Am. Chem. Soc., 2008, 130, 11486-11493; Jewett, et.al. J. Am. Chem. Soc., 2010, 132, 3688-3690; Ning, et. al. Angew. Chem.Int. Ed., 2008, 47, 2253-2255), difluoro-oxanorbornene derivatives (vanBerkel, et. al. ChemBioChem, 2007, 8, 1504-1508), or nitrile oxidederivatives (Lutz, et. al. Macromolecules, 2009, 42, 5411-5413). Incertain embodiments a metal-free click chemistry reaction is ametal-free [3+2] cycloaddition reaction, Diels-Alder reaction, orthiol-alkene radical addition reaction. Exemplary click chemistryreactions and click chemistry groups are described in, e.g., JoergLahann, Click Chemistry for Biotechnology and Materials Science, 2009,John Wiley & Sons Ltd, ISBN 978-0-470-69970-6; Becer, Hoogenboom, andSchubert, Click Chemistry beyond Metal-Catalyzed Cycloaddition,Angewandte Chemie International Edition (2009) 48: 4900-4908. In certainembodiments a click chemistry group comprises a diarylcyclooctyne.

Certain examples of metal free click chemistry are shown in the schemebelow.

Certain metal-free click moieties are known in the literature. Examplesinclude 4-dibenzocyclooctynol (DIBO)

(from Ning et. al; Angew Chem Int Ed, 2008, 47, 2253); difluorinatedcyclooctynes (DIFO or DFO)

(from Codelli, et. al.; J. Am. Chem. Soc. 2008, 130, 11486-11493.);biarylazacyclooctynone (BARAC)

(from Jewett et. al.; J. Am. Chem. Soc. 2010, 132, 3688); orbicyclononyne (BCN)

(From Dommerholt, et. al.; Angew Chem Int Ed, 2010, 49, 9422-9425) ordibenzylcyclooctyne (DBCO)

A reaction scheme involving reaction of DBCO and an azide is shownbelow:

In the above scheme, in various embodiments, A may comprise or consistof a compstatin analog moiety and B may comprise or consist of a CRM,e.g., a polymer, such as a PEG or a POZ or a polypeptide, or B maycomprise or consist of a compstatin analog moiety and A may comprise orconsist of a CRM, e.g., a polymer, such as a PEG or a POZ or apolypeptide.

In some embodiments, the “metal free click-functionalized” moiety is anacetylene or an acetylene derivative which is capable of undergoing[3+2] cycloaddition reactions with complementary azide-bearing moleculesand biomolecules without the use of a metal catalyst.

In some embodiments, the R and R′ groups of the metal-free clickchemistry reagents are a compstatin analog or any molecule describedherein to which a compstatin analog may be conjugated. In someembodiments, such compstatin analogs bear a click-functionalized moietyon a lysine side chain. In some embodiments, such compstatin analogs areconnected to a click-functionalized moiety via a linker. In someembodiments, such compstatin analogs are connected to aclick-functionalized moiety via AEEAc.

In some embodiments, a click chemistry reagent comprises DBCO. Exemplaryreagents and exemplary uses thereof are set forth below:

DBCO-Acid. In some embodiments a DBCO-Acid may be used to react with anamine-containing moiety.

DBCO-NHS ester (above) or DBCO-sulfo-NHS ester (below) may be used toincorporate a DBCO functionality into an amine-containing molecule, suchas a compstatin analog or a polypeptide comprising a lysine residue.

DBCO-PEG4-NHS ester. In some embodiments such reagent is useful forintroducing a DBCO moiety by reaction with an available aminefunctionality. In some aspects, the presence of a PEG chain as ahydrophilic spacer may be useful to, e.g., increase solubility orprovide flexibility.

DBCO-Amine. In some embodiments a click chemistry reagent comprises acarbonyl/carboxyl reactive dibenzylcyclooctyne, which may react withacids, active esters and/or aldehydes.

In certain embodiments a click chemistry reaction involves a cyclooctynedepicted below:

In certain embodiments click chemistry reactions comprise reactionsbetween nitrones and cyclooctynes (see, e.g., Ning, Xinghai; Temming,Rinske P.; Dommerholt, Jan; Guo, Jun; Ania, Daniel B.; Debets, MarjokeF.; Wolfert, Margreet A.; Boons, Geert-Jan et al. (2010). “ProteinModification by Strain-Promoted Alkyne-Nitrone Cycloaddition”.Angewandte Chemie International Edition 49 (17): 3065), oxime/hydrazoneformation from aldehydes and ketones, tetrazine ligations (see, e.g.,Blackman, Melissa L.; Royzen, Maksim; Fox, Joseph M. (2008). “TheTetrazine Ligation: Fast Bioconjugation based on Inverse-electron-demandDiels-Alder Reactivity”. Journal of the American Chemical Society 130(41): 13518-9), tetrazole ligations, the isonitrile-based click reaction(see, e.g., Stackmann, Henning; Neves, AndrÃ© A.; Stairs, Shaun;Brindle, Kevin M.; Leeper, Finian J. (2011). “Exploring isonitrile-basedclick chemistry for ligation with biomolecules”. Organic & BiomolecularChemistry 9 (21): 7303), and the quadricyclane ligation (see, e.g.,Sletten, Ellen M.; Bertozzi, Carolyn R. (2011). “A BioorthogonalQuadricyclane Ligation”. Journal of the American Chemical Society 133(44): 17570-3). In certain embodiments a click chemistry reaction is aStaudinger ligation (phosphine-azide).

Any compstatin analog may be modified to incorporate a click chemistrygroup in various embodiments. For example, a compstatin analogcomprising the sequence of any of SEQ ID NOs: 3-36, 37, 71, 72, 73, or74 may be so modified. In some embodiments any such sequence furthercomprises a lysine residue or an AEEAc-Lys moiety, e.g., at theC-terminus. In some embodiments a click chemistry group is incorporatedafter peptide synthesis. For example, a Lys side chain may be reactedwith azido acetic acid in order to introduce an azide moiety as a clickchemistry group. In some embodiments a click chemistry group isincorporated after cyclization and, in some embodiments, after additionof a blocking moiety at the N- and/or C-terminus. In some embodiments aclick chemistry group is incorporated during peptide synthesis. Forexample, an amino acid comprising a side chain that comprises a clickchemistry group may be used in the synthesis of a compstatin analog. Avariety of such amino acids are commercially available from a number ofsources, e.g., AAPPTec (Louisville, Ky.), Jena Bioscience GmBH (Jena,Germany). In some aspects, methods of making a click chemistryfunctionalized compstatin analog are provided herein.

In some embodiments compositions comprising a compstatin analog and aclick chemistry reagent are provided. The click chemistry reagent may beany molecule capable of reacting with an amino acid side chain orterminus of a compound comprising a compstatin analog so as to install aclick chemistry group, e.g., any click chemistry group known in the art.In some aspects, the composition may be incubated under suitableconditions (which may include providing a suitable catalyst, light(e.g., UV)) to functionalize the compstatin analog with a clickchemistry functionality. In some embodiments, the invention providescompstatin analogs that comprise any click chemistry group including,but not limited to, those described herein. In some embodiments methodsof making a long-acting compstatin analog are provided. In someembodiments the methods comprise mixing a compstatin analog comprising afirst click chemistry group with a CRM comprising a complementary clickchemistry group under conditions suitable for a click chemistry reactionto occur. Additional steps may comprise purifying the resultingconjugate. In some embodiments purifying comprises removing at leastsome unreacted components, e.g., with an appropriate scavenger.

In some embodiments a click chemistry reaction is used to join two ormore CRMs, at least two of which have a compstatin analog moietyattached thereto. The compstatin analog moieties may be the same ordifferent in various embodiments. The compstatin analog moieties may ormay not be attached to the CRM via a click chemistry reaction. Forexample, in some embodiments a first heterobifunctional PEG comprising afirst click chemistry group at a first terminus and an NHS ester at asecond terminus is coupled to a compstatin analog moiety via the NHSester. In a separate reaction, a second heterobifunctional PEGcomprising a second click chemistry group at a first terminus and an NHSester at a second terminus is coupled to a compstatin analog moiety viathe NHS ester. The resulting two compounds are then reacted via a clickchemistry reaction to form a larger molecule comprising two compstatinanalog moieties. PEG is mentioned as an example of a CRM but it shouldbe understood that this approach may be used with any CRM. For example,in some embodiments it may be used with a CRM comprising a polypeptide,e.g., HSA or a portion thereof, or an albumin or albumin-bindingpeptide, or an antibody or portion thereof. In some embodiments thisapproach may be used with a POZ.

Compstatin analogs comprising a click chemistry group have a variety ofuses. In some embodiments a compstatin analog comprising a first clickchemistry group is reacted with any entity that comprises acomplementary click chemistry group. The entity comprising thecomplementary click chemistry group may comprise, for example, a label(e.g., a fluorophore, fluorescent protein, radioisotope, etc.), anaffinity reagent, an antibody, a targeting moiety, a metal, a particle,etc. In some embodiments a click chemistry group is used to attach acompstatin analog moiety to a surface, wherein the surface comprises oris functionalized to comprise a complementary click chemistry group. Insome embodiments a surface is for a sensor, e.g., a surface or sensorfor capture/detection of C3. In some embodiments a surface forms part ofa medical device, tubing, membrane, reservoir, implant, or othermaterial that may come in contact with blood (e.g., extracorporeally) orbe temporarily or indefinitely implanted into the body of a subject(e.g., a prosthetic device or drug delivery device). In some embodimentsa surface is functionalized with compstatin analog to reduce complementactivation thereon. In some embodiments a device or tubing is used forcirculating blood, e.g., for dialysis, during surgery, etc. In someembodiments a device is a hemodialyzer or an extracorporeal circulatorysupport unit. Such compstatin analog functionalized devices and methodsof making thereof are provided herein.

In some embodiments of the invention, a compstatin analog comprises botha cell-reactive functional group and a CRM. In some aspects, theinvention provides variants of the molecules of any of theafore-mentioned cell-reactive compstatin analogs wherein a cell-reactivefunctional group or moiety is replaced by a (CH₂CH₂O)_(n) moiety havinga molecular weight of at least 500 daltons, e.g., at least 1,500 daltonsup to about 100,000 daltons (e.g., an average molecular weight of about20,000; 30,000; 40,000; 50,000; 60,000; 70,000; 80,000; 90,000; or100,000 daltons). In some embodiments the average molecular weight ofthe compound or (CH₂CH₂O)_(n) moieties is at least 20,000 daltons, up toabout 100,000; 120,000; 140,000; 160,000; 180,000; or 200,000 daltons.

Exemplary long-acting compstatin analogs are set forth below, wherein nis sufficient to provide an average molecular weight of between about500; 1,000; 1,500; 2,000; 5,000; 10,000; 20,000; 30,000; 40,000; 50,000;60,000; 70,000; 80,000; 90,000; and 100,000 daltons. In some embodimentsn is sufficient to provide an average molecular weight of between about20,000 daltons, up to about 100,000; 120,000; 140,000; 160,000; 180,000;or 200,000 daltons.

(SEQ ID NO: 58) (CH₂CH₂O)_(n)C(═O)-Ile-Cys-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys- Thr-NH₂) (SEQ ID NO: 59)Ac-Ile-Cys*-Val-(1Me)Trp-Gln-Asp- Trp-Gly-Ala-His-Arg-Cys*-Thr-NH-CH₂CH₂OCH₂CH₂OCH₂—C(═O)-Lys-C(═O)—(CH₂CH₂O)n—NH₂ (SEQ ID NO: 60)Ac-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-Lys-C(═O)—(CH₂CH₂O)n—NH₂. (SEQ ID NO: 61)Ac-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-(Gly)₅-Lys- C(═O)—(CH₂CH₂O)n—NH₂(SEQ ID NO: 62) Ac-(CH₂CH₂O)nC(═O)Lys-(Gly)5-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly- Ala-His-Arg-Cys*-Thr-NH₂)(SEQ ID NO: 63) Ac-(CH₂CH₂O)nC(═O)Lys-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His- Arg-Cys*-Thr-NH₂)

In SEQ ID NO: 58, the (CH₂CH₂O)n is coupled via an amide bond to theN-terminal amino acid. In SEQ ID NOs: 59-63, the (CH₂CH₂O)n moiety iscoupled via an amide bond to a Lys side chain; thus it will beunderstood that the NH₂ at the C-terminus in SEQ ID NOs: 59, 60, and 61,represents amidation of the C-terminus of the peptide, and it will beunderstood that in SEQ ID NOs: 62 and 63, the Ac at the N-terminusrepresents acetylation of the N-terminus of the peptide, as describedabove. It will also be appreciated by those of ordinary skill in the artthat a free end of a (CH₂CH₂O)_(n) moiety typically terminates with an(OR) where the underlined O represents the O atom in the terminal(CH₂CH₂O) group. (OR) is often a moiety such as a hydroxyl (OH) ormethoxy (—OCH₃) group though other groups (e.g., other alkoxy groups)could be used. Thus SEQ ID NO: 59, for example, may be represented as

(SEQ ID NO: 64) Ac-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-NH-CH₂CH₂OCH₂CH₂OCH₂- C(═O)-Lys-(C(═O)—(CH₂CH₂O)_(n)—R)—NH2wherein R is, e.g., either H or CH₃ in the case of a linear PEG. In thecase of a bifunctional, branched or star-shaped PEG, R represents theremainder of the molecule. Further, it will be understood that themoiety comprising the reactive functional group may vary, as describedherein (e.g., according to any of the formulas described herein). Forexample, long-acting compstatin analogs comprising the same peptidesequence as SEQ ID NO: 64, in which the moiety comprising the reactivefunctional group comprises an ester and/or alkyl chain may berepresented as follows

(SEQ ID NO: 65) Ac-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-NH—CH₂CH₂OCH₂CH₂OCH₂—C(═O)-Lys-(C(═O)—(CH2)_(m)—(CH₂CH₂O)_(n)—R)—NH2; (SEQ ID NO: 66)Ac-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-NH—CH₂CH₂OCH₂CH₂OCH₂—C(═O)-Lys-(C(═O)—(CH₂)_(m)C(═O)—(CH₂CH₂O)_(n)—R)—NH2 (SEQ ID NO: 67)Ac-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-NH—CH₂CH₂OCH₂CH₂OCH₂—C(═O)-Lys-(C(═O)—(CH₂)_(m—)C(═O)—(CH₂)j (CH₂CH₂O)_(n)—R)—NH2In SEQ ID NOs: 65-67 m may range from 1 up to about 2, 3, 4, 5, 6, 7, 8,10, 15, 20, or 30 in various embodiments, In SEQ ID NOs: 67 j may rangefrom 1 up to about 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, or 30 in variousembodiments.It will also be appreciated that, as described herein, in variousembodiments other moieties may be incorporated between the Lys-(C(═O)—and (CH₂CH₂O)_(n)—R, such as an amide, aromatic ring (e.g., asubstituted or unsubstituted phenyl), or a substituted or unsubstitutedcycloalkyl structure.

The invention provides variants of SEQ ID NOs: 58-67 in which

(SEQ ID NO: 75) -Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-is replaced by an amino acid sequence comprising the amino acid sequenceof any other compstatin analog, e.g., of any of SEQ ID NOs 3-27 or29-36, 37, 71, 72, 73, or 74 with the proviso that blocking moiet(ies)present at the N- and/or C-termini of a compstatin analog may be absent,replaced by a linker (which may comprise a blocking moiety), or attachedto a different N- or C-terminal amino acid present in the correspondingvariant(s).

Any compstatin analog, e.g., any compound comprising any of SEQ ID NOs:3-37, 71, 72, 73, or 74 may, in various embodiments, can be attached viaor near its N-terminal or C-terminal end (e.g., via a side chain of anamino acid at or near its N-terminal or C-terminal amino acid) directlyor indirectly to any moiety comprising a reactive functional group,e.g., any compound of Formulae I-XVI or Formulae A-H.

In some embodiments the CRM comprises a polypeptide that occurs in humanserum, or a fragment thereof or a substantially similar variant of thepolypeptide or fragment thereof. In some embodiments the polypeptide,fragment, or variant has a molecular weight of between 5 kD and 150 kD,e.g., at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 kd, or more,e.g., between 100 and 120, or 120 and 150 kD. In some embodiments,producing a long-acting compstatin analog comprises reacting acompstatin analog comprising a reactive functional group with one ormore amino acid side chains of the polypeptide, wherein the side chaincomprises a compatible functional group. In some embodiments, producinga long-acting compstatin analog comprises reacting a compstatin analogcomprising a reactive functional group with the N-terminal amine and/orC-terminal carboxyl group of the polypeptide. In some embodiments,producing a long-acting compstatin analog comprises reacting acompstatin analog comprising an amine-reactive functional group withamino acids having a side chain comprising a primary amine (e.g.,lysine) and/or with the N-terminal amine of the polypeptide. In someembodiments, producing a long-acting compstatin analog comprisesreacting a compstatin analog comprising a carboxyl-reactive functionalgroup with the C-terminal carboxyl group of the polypeptide. In someembodiments a compstatin analog moiety is attached at each terminus ofthe polypeptide and, optionally, to the side chain of one or moreinternal amino acids. In some embodiments, producing a long-actingcompstatin analog comprises reacting a compstatin analog comprising asulfhydryl-reactive functional group with one or more sulfhydryl groupsof the polypeptide.

In some embodiments, at least one reactive functional group isintroduced into the polypeptide. For example, in some embodiments atleast one side chain of the polypeptide is modified to convert a firstreactive functional group to a different reactive functional group priorto reaction with the compstatin analog. In some embodiments a thiol isintroduced. Several methods are available for introducing thiols intobiomolecules, including the reduction of intrinsic disulfides, as wellas the conversion of amine, aldehyde or carboxylic acid groups to thiolgroups. Disulfide crosslinks of cystines in proteins can be reduced tocysteine residues by dithiothreitol (DTT),tris-(2-carboxyethyl)phosphine (TCEP), or tris-(2-cyanoethyl)phosphine.Amines can be indirectly thiolated by reaction with succinimidyl3-(2-pyridyldithio)propionate (SPDP) followed by reduction of the3-(2-pyridyldithio)propionyl conjugate with DTT or TCEP. Amines can beindirectly thiolated by reaction with succinimidyl acetylthioacetatefollowed by removal of the acetyl group with 50 mM hydroxylamine orhydrazine at near-neutral pH. Amines can be directly thiolated byreaction with 2-iminothiolane, which preserve the overall charge of themolecule and introduces a free thiol. Tryptophan residues in thiol-freeproteins can be oxidized to mercaptotryptophan residues, which can thenbe modified by iodoacetamides or maleimides. A polypeptide comprisingone or more thiols may be reacted with a compstatin analog comprising amaleimide group, such as

(SEQ ID NO: 68) Ac-Ile-Cys*-Val-Trp(1-Me)-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-AEEAc-Lys-(C(═O)—CH₂)₅-Mal)-NH₂to generate a long-acting compstatin analog.

In some embodiments the polypeptide is recombinantly produced. In someembodiments the polypeptide is at least in part recombinantly produced(e.g., in bacteria or in eukaryotic host cells such as fungal, insect,plant, or vertebrate) and/or at least in part produced using chemicalsynthesis. In some embodiments the polypeptide is purified. For example,in some embodiments the polypeptide is purified from a host cell lysateor from culture medium into which it has been secreted by host cells. Insome embodiments the polypeptide is glycosylated. In some embodimentsthe polypeptide is non-glycosylated. In some embodiments the polypeptideis human serum albumin (HSA). In some embodiments a substantiallysimilar variant of the polypeptide is sufficiently similar to thepolypeptide of which it is a variant so as to not be recognized asforeign by a normal immune system of a subject, e.g., a human subject.In some embodiments alterations in the sequence of substantially similarvariant as compared with the polypeptide of which it is a variant areselected so as to avoid generating MHC Class I epitopes. Various methodsknown in the art can be used to predict whether a sequence comprises anMHC Class I epitope.

In some embodiments, one or more amino acids in a polypeptide or linkeror composition may be selected to be hydrophobic or hydrophilic orselected to confer increased hydrophilicity or, in some embodiments,increased hydrophobicity, on a compound that contains it. As known inthe art, the terms “hydrophilic” and “hydrophobic” are used to refer tothe degree of affinity that a substance has with water. In some aspectsa hydrophilic substance has a strong affinity for water, tending todissolve in, mix with, or be wetted by water, while a hydrophobicsubstance substantially lacks affinity for water, tending to repel andnot absorb water and tending not to dissolve in or mix with or be wettedby water. Amino acids can be classified based on their hydrophobicity aswell known in the art. Examples of “hydrophilic amino acids” arearginine, lysine, threonine, alanine, asparagine, glutamine, aspartate,glutamate, serine, and glycine. Examples of “hydrophobic amino acids”are tryptophan, tyrosine, phenylalanine, methionine, leucine,isoleucine, and valine. In certain embodiments an analog of a standardamino acid is used, wherein the analog has increased or decreasedhydrophilic or hydrophobic character as compared with the amino acid ofwhich it is an analog.

The invention further provides multimers, e.g., concatamers, comprisingtwo or more (e.g., between 2 and 10) compstatin analogs comprising aCRM, wherein the average molecular weight of the resulting molecule (orthe CRM components thereof) is between 20,000; 30,000; 40,000; 50,000;60,000; 70,000; 80,000; 90,000; and 100,000 daltons. In some embodimentsthe average molecular weight of the resulting molecule (or the CRMcomponents thereof) is at least 20,000 daltons, up to about 100,000;120,000; 140,000; 160,000; 180,000; or 200,000 daltons. In someembodiments, the compstatin analogs comprising a CRM can be linked usingany of the linking moieties described above. Compositions and methodsfor making long-acting compstatin analogs, and intermediates in thesynthesis, are aspects of the invention.

A wide variety of methods and assays useful for detection of polymers,e.g., PEGs, POZs, and/or polypeptides and/or useful for measurement ofphysical and/or structural properties of polymers, e.g., PEGs, POZs,and/or polypeptides are known in the art and may, if desired, be used todetect a compstatin analog, e.g., a cell-reactive, long-acting, targetedcompstatin analog or a compstatin analog moiety. For example, methodsand assays useful for determining properties such as aggregation,solubility, size, structure, melting properties, purity, presence ofdegradation products or contaminants, water content, hydrodynamicradius, etc., are available. Such methods include, e.g., analyticalcentrifugation, various types of chromatography such as liquidchromatography (e.g., HPLC-ion exchange, HPLC-size exclusion,HPLC-reverse phase), light scattering, capillary electrophoresis,circular dichroism, isothermal calorimetry, differential scanningcalorimetry, fluorescence, infrared (IR), nuclear magnetic resonance(NMR), Raman spectroscopy, refractometry, UV/Visible spectroscopy, massspectrometry, immunological methods, etc. It will be understood thatmethods may be combined. In some aspects, a cell-reactive, long-acting,or targeted compstatin analog (or composition comprising acell-reactive, long-acting, or targeted compstatin analog) has one ormore properties described herein, as assessed using any of the foregoingmethods. In some aspects, methods useful to detect and/or quantify along-acting compstatin analog are described herein.

VII. Targeted Compstatin Analogs

The invention provides targeted compstatin analogs that comprise atargeting moiety and a compstatin analog moiety, wherein the targetingmoiety binds non-covalently to a target molecule. In some aspects, theinvention provides targeted compstatin analogs analogous to thecell-reactive compstatin analogs described in Section VI, wherein thecompounds comprise a targeting moiety in addition to, or instead of, acell-reactive moiety. The targeting moiety can comprise, e.g., anantibody, polypeptide, peptide, nucleic acid (e.g., an aptamer),carbohydrate, small molecule, or supramolecular complex, thatspecifically binds to the target molecule. In some embodiments, theaffinity (as measured by the equilibrium dissociation constant, Kd) oftargeting moiety for the target molecule (as measured by the equilibriumdissociation constant, Kd) is 10⁻³ M or less, e.g., 10⁻⁴ M or less,e.g., 10⁻⁵ M or less, e.g., 10⁻⁶M or less, 10⁻⁷M or less, 10⁻⁸M or less,or 10⁻⁹M or less under the conditions tested, e.g., under physiologicalconditions.

In those embodiments of the invention in which the targeting moiety isan antibody, the antibody may be any immunoglobulin or a derivativethereof, which maintains binding ability, or any protein having abinding domain which is homologous or largely homologous to animmunoglobulin binding domain. Such proteins may be derived from naturalsources, or partly or wholly synthetically produced (e.g., usingrecombinant DNA techniques, chemical synthesis, etc.). The antibody canbe of any species, e.g., human, rodent, rabbit, goat, chicken, etc. Theantibody may be a member of any immunoglobulin class, including any ofthe human classes: IgG, IgM, IgA, IgD, and IgE. In various embodimentsof the invention the antibody may be a fragment of an antibody such asan Fab′, F(ab′).sub.2, scFv (single-chain variable) or other fragmentthat retains an antigen binding site, or a recombinantly produced scFvfragment, including recombinantly produced fragments. See, e.g., Allen,T., Nature Reviews Cancer, Vol. 2, 750-765, 2002, and referencestherein. Monovalent, bivalent or multivalent antibodies can be used. Theantibody may be a chimeric antibody in which, for example, a variabledomain of rodent origin is fused to a constant domain of human origin,thus retaining the specificity of the rodent antibody. In someembodiments, a human antibody or portion thereof is generated, forexample, in rodents whose genome incorporates human immunoglobulingenes, using a display technology such as phage display, etc. In someembodiments, a humanized antibody is generated by grafting one or morecomplementarity determining region(s) from a non-human species (e.g.,mouse) into a human antibody sequence. The antibody may be partially orcompletely humanized. See, e.g., Almagro J C, Fransson J. Humanizationof antibodies. Front Biosci. 13:1619-33 (2008) for review of variousmethods of obtaining humanized antibodies that may be used to obtain atargeting moiety of use in the invention. An antibody may be polyclonalor monoclonal, though for purposes of the present invention monoclonalantibodies are generally preferred. In certain embodiments of theinvention a F(ab′)2 or F(ab′) fragment is use while in other embodimentsantibodies comprising an Fc domain are used. Methods for producingantibodies that specifically bind to virtually any molecule of interestare known in the art. For example, monoclonal or polyclonal antibodiescan be purified from natural sources, e.g., from blood or ascites fluidof an animal that produces the antibody (e.g., following immunizationwith the molecule or an antigenic fragment thereof) or can be producedrecombinantly, in cell culture. Methods of generating antibodyfragments, e.g., by digestion, disulfide reduction, or synthesis areknown in the art.

In various embodiments of the invention a targeting moiety can be anymolecule that specifically binds to a target molecule through amechanism other than an antigen-antibody interaction. Such a targetingmoiety is referred to as a “ligand”. For example, in various embodimentsof the invention a ligand can be a polypeptide, peptide, nucleic acid(e.g., DNA or RNA), carbohydrate, lipid or phospholipid, or smallmolecule. In some embodiments a small molecule is an organic compound,whether naturally-occurring or artificially created, that has relativelylow molecular weight and is not a protein, polypeptide, nucleic acid, orlipid, typically with a molecular weight of less than about 1500 g/moland typically having multiple carbon-carbon bonds. In general, anaptamer is an oligonucleotide (e.g., RNA or DNA, optionally comprisingone or more modified nucleosides (e.g., bases or sugars other than the 5standard bases (A, G, C, T, U) or sugars (ribose and deoxyribose) foundmost commonly in RNA and DNA), or modified internucleoside linkages(e.g., non-phosphodiester bonds) that, e.g., stabilize the molecule,e.g., by rendering it more resistant to degradation by nucleases) thatbinds to a particular protein. In some embodiments an oligonucleotide isup to about 100 nucleosides long, e.g., between 12 and 100 nucleosideslong. Aptamers can be derived using an in vitro evolution process calledSELEX, and methods for obtaining aptamers specific for a protein ofinterest are known in the art. See, e.g., Brody E N, Gold L. JBiotechnol. 2000 March; 74(1):5-13. In some embodiments, a peptidenucleic acid or locked nucleic acid is used.

In certain embodiments of the invention a targeting moiety comprises apeptide. In some embodiments a peptide that binds to a target moleculeof interest is identified using a display technology such as phagedisplay, ribosome display, yeast display, etc.

Small molecules can be used as ligands. Methods for identifying suchligands are known in the art. For example in vitro screening of smallmolecule libraries, including combinatorial libraries, andcomputer-based screening, e.g., to identify small organic compounds thatbind to concave surfaces (pockets) of proteins, can identify smallmolecule ligands for numerous proteins of interest (Huang, Z., Pharm. &Ther. 86: 201-215, 2000).

In certain embodiments of the invention targeting moieties are notproteins or molecules that are typically used as carriers and conjugatedto antigens for the purpose of raising antibodies. Examples are carrierproteins or molecules such as bovine serum albumin, keyhole limpethemocyanin, bovine gamma globulin, and diphtheria toxin. In certainembodiments of the invention the targeting moiety is not an Fc portionof an immunoglobulin molecule. In some embodiments, a targeting moietyis part of a complex comprising one or more additional moieties to whichit is covalently or noncovalently attached.

In various embodiments of the invention a target molecule can be anymolecule produced by a cell (including any forms expressed on the cellsurface or modified forms thereof resulting at least in part fromextracellular modification). In some embodiments a target molecule is anextracellular substance present in or on a tissue. In some embodiments,a target molecule is characteristic of a particular diseased orphysiological state or characteristic of one or more cell type(s) ortissue type(s). A target molecule is often a molecule at least partlypresent at the cell surface (e.g., a transmembrane or otherwisemembrane-attached protein) so that at least a portion of the molecule isaccessible to binding by an extracellular binding agent such as anantibody. A target molecule may, but need not be, cell type specific.For example, a cell type specific target molecule is often a protein,peptide, mRNA, lipid, or carbohydrate that is present at a higher levelon or in a particular cell type or cell type(s) than on or in many othercell types. In some instances a cell type specific target molecule ispresent at detectable levels only on or in a particular cell type ofinterest. However, it will be appreciated that a useful cell typespecific target molecule need not be absolutely specific for the celltype of interest in order to be considered cell type specific. In someembodiments, a cell type specific target molecule for a particular celltype is expressed at levels at least 3 fold greater in that cell typethan in a reference population of cells which may consist, for example,of a mixture containing cells from a plurality (e.g., 5-10 or more) ofdifferent tissues or organs in approximately equal amounts. In someembodiments, the cell type specific target molecule is present at levelsat least 4-5 fold, between 5-10 fold, or more than 10-fold greater thanits average expression in a reference population. In some embodiments,detection or measurement of a cell type specific target molecule allowsone of ordinary skill in the art to distinguish a cell type or types ofinterest from cells of many, most, or all other types. In general, thepresence and/or abundance of most target molecules may be determinedusing one or more standard techniques such as Northern blotting, in situhybridization, RT-PCR, sequencing, immunological methods such asimmunoblotting, immunodetection (e.g., by immunohistochemistry), orfluorescence detection following staining with fluorescently labeledantibodies (e.g., using FACS), oligonucleotide or cDNA microarray ormembrane array, protein microarray analysis, mass spectrometry, etc.

In some embodiments, a target molecule is a channel, transporter,receptor, or other molecule at least in part exposed at the cellsurface. In some embodiments a target molecule is an anion transporteror water channel (e.g., an aquaporin protein).

In some embodiments, the target molecule is a protein at least in partexposed at the surface of red blood cells, such as a glycophorin (e.g.,glycophorin A, B, C, or D) or band 3.

In some embodiments, the target molecule is a protein at least in partexposed at the surface of endothelial cells. In some embodiments, thetarget molecule is present at the surface of normal, healthyvasculature. In some embodiments, the target molecule is present at thesurface of activated endothelial cells. In some embodiments, the targetmolecule is present at the surface of activated endothelial cells butnot at the surface of non-activated endothelial cells. In someembodiments a target molecule is a molecule whose expression or exposureis induced by a stimulus such as injury or inflammation. In someembodiments, a target molecule would be recognized as “non-self” by arecipient receiving a transplant containing cells that express thetarget molecule. In some embodiments, the target molecule is acarbohydrate xenoantigen to which antibodies are commonly found in humanbeings. In some embodiments the carbohydrate comprises a blood groupantigen. In some embodiments the carbohydrate comprises a xenoantigen,e.g., an alpha-gal epitope (Galalpha1-3Galbeta1-(3)4GlcNAc-R) (see,e.g., Macher B A and Galili U. The Galalpha1, 3Galbeta1, 4GlcNAc-R(alpha-Gal) epitope: a carbohydrate of unique evolution and clinicalrelevance. Biochim Biophys Acta. 1780(2):75-88 (2008).

In some embodiments of the invention, a compstatin analog comprises botha targeting moiety and a CRM.

In some embodiments, a targeted compstatin analog comprises multipletargeting moieties, which can be the same or different. Differenttargeting moieties may bind to the same target molecule or to differenttarget molecules. The invention provides a targeted compstatin analogthat is multivalent with respect to the targeting moiety, the compstatinanalog, or both.

In general, the invention encompasses any method of producing a compoundcomprising a compstatin analog moiety and a targeting moiety, and theresulting compounds. In some embodiments, a targeted compstatin analogmay be produced using methods generally similar to those described inSection VI, wherein a targeting moiety is used instead of, or inaddition to, a cell-reactive moiety. In some embodiments, a targetedcompstatin analog comprising a peptide as a targeting moiety issynthesized as a polypeptide chain comprising a compstatin analog moietyand a peptide targeting moiety. Optionally, the polypeptide chaincomprises one or more spacer peptides between the compstatin analogmoiety and the targeting moiety.

In some embodiments, a targeted compstatin analog has a molar activityof at least about 10%, 20%, or 30%, e.g., between 30% and 40%, between30% and 50%, between 30% and 60%, between 30% and 70%, between 30% and80%, between 30% and 90%, or more, of the activity of a correspondingcompstatin analog having the same amino acid sequence (and, ifapplicable, one or more blocking moiet(ies)) but not comprising atargeting moiety. In some embodiments wherein a targeted compstatinanalog comprises multiple compstatin analog moieties, the molar activityof the targeted compstatin analog is at least about 10%, 20%, or 30%,e.g., between 30% and 40%, between 30% and 50%, between 30% and 60%,between 30% and 70%, between 30% and 80%, between 30% and 90%, or more,of the sum of the activities of said compstatin analog moieties.Compositions and methods for making targeted compstatin analogs, andintermediates in the synthesis, are aspects of the invention.

VIII. Uses

Cell-reactive, long-acting, or targeted compstatin analogs have a widevariety of uses. Without limiting the invention in any way, certain usesof cell-reactive, long-acting, or targeted compstatin analogs, andrelated aspects of the invention, are described herein. In someembodiments, a cell-reactive, long-acting, or targeted compstatin analogis administered to a subject suffering from or at risk ofcomplement-mediated damage to an organ, tissue, or cells. In someembodiments, a cell-reactive compstatin analog is contacted with anorgan, tissue, or cells ex vivo and become covalently attached thereto.The organ, tissue, or cells are introduced into a subject and areprotected from damage that would otherwise be caused by the recipient'scomplement system.

Compstatin analogs that do not bind covalently to cells can be used forpurposes described herein. For example, a compstatin analog modifiedwith a moiety that increases the lifetime of the compound in the bodyand/or a compstatin analog comprising a moiety that targets thecompstatin analog to a cell type or location susceptible to complementactivation can be used, and the invention encompasses such uses. In someembodiments, a long-acting compstatin analog is used. In someembodiments a compstatin analog comprising a targeting moiety is used.In some embodiments, a compstatin analog comprising both a moiety thatextends the lifetime of the compound in the body and a targeting moietyis used. Where the discussion below refers to a cell-reactive compstatinanalog, the invention provides analogous compositions and methodsrelating to targeted compstatin analogs and (at least in those aspectspertaining to administration of a compstatin analog to a subject)embodiments in which a compstatin analog that does not comprise atargeting moiety or a cell-reactive moiety, optionally a long-actingcompstatin analog, is used instead of, or in addition to, acell-reactive compstatin analog.

Certain uses of interest include: (1) protecting red blood cells (RBCs)from complement-mediated damage in individuals with disorders such asparoxysmal nocturnal hemoglobinuria or atypical hemolytic uremicsyndrome or other disorders characterized by complement-mediated RBClysis; (2) protecting transplanted organs, tissues, and cells fromcomplement-mediated damage; (3) reducing ischemia/reperfusion (I/R)injury (e.g., in individuals suffering from trauma, vascularobstruction, myocardial infarction, or other situations in which I/Rinjury may occur); and (4) protecting various body structures (e.g., theretina) or membranes (e.g., synovial membrane) that may be exposed tocomplement components from complement mediated damage in any of avariety of different complement-mediated disorders. The beneficialeffects of inhibiting complement activation at the surface of cells orother body structures are not limited to those resulting directly fromprotection of the cells or structures themselves against directcomplement-mediated damage (e.g., preventing cell lysis). For example,inhibiting complement activation using a cell-reactive compstatin analogmay reduce the generation of anaphylotoxins and resultinginflux/activation of neutrophils and other pro-inflammatory eventsand/or reduce potentially damaging release of intracellular contents,thereby potentially having beneficial effects on remote organ systems orthroughout the body.

A. Blood Cell Protection

In some embodiments of the invention, a cell-reactive compstatin analog,cell-targeted compstatin analog, and/or non-targeted compstatin analog(e.g., a long-acting non-targeted compstatin analog) is used to protectblood cells against complement-mediated damage. The blood cells may beany cellular component of the blood, e.g., red blood cells (RBCs), whiteblood cells (WBCs), and/or platelets. In some embodiments, acell-targeted compstatin analog is targeted to a target molecule exposedat the cell surface of RBCs such as a glycophorin or band 3. A varietyof disorders are associated with complement-mediated damage to bloodcells. Such disorders can result, for example, from deficiencies ordefects in one or more of an individual's cellular or soluble CRPs,e.g., due to (a) mutation(s) in the gene(s) encoding such proteins; (b)mutation(s) in genes required for production or proper function of oneor more CRPs, and/or (c) presence of autoantibodies to one or more CRPs.Complement-mediated RBC lysis can result from the presence ofautoantibodies against RBC antigens which may arise due to a diverse setof causes (often being idiopathic). Individuals having such mutation(s)in genes encoding CRPs and/or having antibodies against CRPs or againsttheir own RBCs are at increased risk of disorders involvingcomplement-mediated RBC damage. Individuals who have had one or moreepisodes characteristic of a disorder are at increased risk of arecurrence.

Paroxysmal nocturnal hemoglobinuria (PNH) is a relatively rare disordercomprising an acquired hemolytic anemia characterized bycomplement-mediated intravascular hemolysis, hemoglobinuria, bone marrowfailure, and thrombophilia (propensity to develop blood clots). Itaffects an estimated 16 individuals per million worldwide, occurs inboth sexes, and can arise at any age, frequently striking young adults(Bessler, M. & Hiken, J., Hematology Am Soc Hematol Educ Program,104-110 (2008); Hillmen, P. Hematology Am Soc Hematol Educ Program,116-123 (2008)). PNH is a chronic and debilitating disease punctuated byacute hemolytic episodes and results in significant morbidities andreduced life expectancy. In addition to anemia, many patients experienceabdominal pain, dysphagia, erectile dysfunction, and pulmonaryhypertension, and are at increased risk of renal failure andthromboembolic events.

PNH was first described as a distinct entity in the 1800s, but it wasonly in the 1950s, with discovery of the alternative pathway ofcomplement activation, that the cause of hemolysis in PNH was firmlyestablished (Parker C J. Paroxysmal nocturnal hemoglobinuria: anhistorical overview. Hematology Am Soc Hematol Educ Program. 93-103(2008)). CD55 and CD59 are normally attached to the cell membrane viaglycosyl phosphatidylinositol (GPI) anchors (glycolipid structures thatanchor certain proteins to the plasma membrane). PNH arises as aconsequence of nonmalignant clonal expansion of hematopoietic stemcell(s) that have acquired a somatic mutation in the PIGA gene, whichencodes a protein involved in synthesis of GPI anchors (Takeda J, et al.Deficiency of the GPI anchor caused by a somatic mutation of the PIG-Agene in paroxysmal nocturnal hemoglobinuria. Cell. 73:703-711 (1993)).Progeny of such stem cells are deficient in GPI-anchored proteins,including CD55 and CD59. This defect renders these cells susceptible tocomplement-mediated RBC lysis. Flow cytometric analysis using antibodiesto GPI-anchored proteins is often used for diagnosis. It detectsdeficiency of GPI-anchored proteins at the cell surface and allowsdetermination of the degree of deficiency and the proportion of affectedcells (Brodsky R A. Advances in the diagnosis and therapy of paroxysmalnocturnal hemoglobinuria. Blood Rev. 22(2):65-74 (2008). PNH type IIIRBCs are completely deficient in GPI-linked proteins and are highlysensitive to complement whereas PNH type II RBCs have a partialdeficiency and are less sensitive. FLAER is a fluorescently labeledinactive variant of proaerolysin (a bacterial toxin that binds GPIanchors) and is increasingly used together with flow cytometry fordiagnosis of PNH. Lack of binding of FLAER to granulocytes is sufficientfor diagnosis of PNH. In some embodiments, a cell-reactive compstatinanalog protects PNH RBCs from deposition of C3b.

In some embodiments, a cell-reactive, long-acting, or targetedcompstatin analog is administered to a subject suffering from atypicalhemolytic syndrome (aHUS). aHUS is a chronic disorder characterized bymicroangiopathic hemolytic anemia, thrombocytopenia, and acute renalfailure and is caused by inappropriate complement activation, often dueto mutations in genes encoding complement regulatory proteins(Warwicker, P., et al. Kidney Int 53, 836-844 (1998); Kavanagh, D. &Goodship, T. Pediatr Nephrol 25, 2431-2442 (2010). Mutations in thecomplement factor H (CFH) gene are the most common genetic abnormalityin patients with aHUS, and 60-70% of these patients die or reach endstage renal failure within one year after disease onset (Kavanagh &Goodship, supra.) Mutations in factor I, factor B, C3, factor H-relatedproteins 1-5, and thrombomodulin have also been described. Other causesof aHUS include autoantibodies against complement regulatory proteinssuch as CFH. In some embodiments, a cell-reactive, long-acting, ortargeted compstatin analog is administered to a subject that has beenidentified as having a mutation in factor I, factor B, C3, factorH-related proteins 1-5, or thrombomodulin or has been identified ashaving antibodies against a complement regulatory protein, e.g., CFH.

Complement-mediated hemolysis occurs in a diverse group of otherconditions including autoimmune hemolytic anemias that involveantibodies that bind to RBCs and lead to complement-mediated hemolysis.For example, such hemolysis can occur in primary chronic cold agglutinindisease and certain reactions to drugs and other foreign substances(Berentsen, S., et al., Hematology 12, 361-370 (2007); Rosse, W. F.,Hillmen, P. & Schreiber, A. D. Hematology Am Soc Hematol Educ Program,48-62 (2004)). In some embodiments of the invention a cell-reactivecompstatin analog is administered to a subject suffering from or at riskof chronic cold agglutinin disease. In another embodiment, acell-reactive compstatin analog is used to treat a subject sufferingfrom or at risk of the HELLP syndrome, which is defined by the existenceof hemolysis, elevated liver enzymes, and low platelet count and isassociated with mutations in complement regulatory protein(s) in atleast some subjects (Fakhouri, F., et al., 112: 4542-4545 (2008)).

In other embodiments, cell-reactive compstatin analogs are used toprotect RBCs or other cellular components of blood to be transfused intoa subject. Certain examples of such uses are discussed further in below.As noted above, targeted and/or long-acting compstatin analogs can beused in the above methods for inhibiting complement-mediated hemolysisand/or RBC damage. In some embodiments, a long-acting compstatin analogcomprising a (CH2CH2O) moiety is used to treat PNH or aHUS.

B. Transplantation

Transplantation is a therapeutic approach of increasing importance,providing a means to replace organs and tissues that have been damagedthrough trauma, disease, or other conditions. Kidneys, liver, lungs,pancreas, and heart are among the organs that can be successfullytransplanted. Tissues that are frequently transplanted include bones,cartilage, tendons, cornea, skin, heart valves, and blood vessels.Pancreatic islet or islet cell transplantation is a promising approachfor treatment of diabetes, e.g., type I diabetes. For purposes of theinvention, an organ, tissue, or cell (or population of cells) that is betransplanted, is being transplanted, or has been transplanted may bereferred to as a “graft”. For purposes hereof, a blood transfusion isconsidered a “graft”.

Transplantation subjects the graft to a variety of damaging events andstimuli that can contribute to graft dysfunction and, potentially,failure. For example, ischemia-reperfusion (I/R) injury is a common andsignificant cause of morbidity and mortality in the case of many grafts(particularly solid organs) and can be a major determinant of likelihoodof graft survival. Transplant rejection is one of the major risksassociated with transplants between genetically different individualsand can lead to graft failure and a need to remove the graft from therecipient.

In some embodiments of the invention, a cell-reactive compstatin analog,cell-targeted compstatin analog, and/or a long-acting compstatin analogis used to protect a graft from complement-mediated damage. Acell-reactive compstatin analog reacts with cells of the graft, becomescovalently attached thereto, and inhibits complement activation. Acell-targeted compstatin analog binds to a target molecule in the graft(e.g., expressed by endothelial cells or other cells in the graft) andinhibits complement activation. A target molecule may be, e.g., is amolecule whose expression is induced or stimulated by a stimulus such asinjury or inflammation, molecule that would be recognized as “non-self”by the recipient, a carbohydrate xenoantigen to which antibodies arecommonly found in human beings such as a blood group antigen or axenoantigen, e.g., a molecule comprising an alpha-gal epitope. In someembodiments, a reduction in complement activation can be demonstrated bya reduction in average C4d deposition in blood vessels of grafts thathave been contacted with a compstatin analog, e.g., a cell-reactivecompstatin analog, as compared with the average level of C4d depositionin grafts that have not been contacted with a compstatin analog (e.g.,in subjects who are matched with respect to the grafts and other therapythat they receive).

A graft can be contacted with a cell-reactive, long-acting, or targetedcompstatin analog prior to, during, and/or after being transplanted, invarious embodiments of the invention. For example, prior totransplantation a graft removed from a donor can be contacted with aliquid comprising a cell-reactive, long-acting, or targeted compstatinanalog. For example, the graft can be bathed in and/or perfused with thesolution. In another embodiment, a cell-reactive, long-acting, ortargeted compstatin analog is administered to a donor prior to removalof the graft. In some embodiments, a cell-reactive, long-acting, ortargeted compstatin analog is administered to a recipient during and/orafter the introduction of the graft. In some embodiments, acell-reactive compstatin, long-acting, or targeted analog is deliveredlocally to the transplanted graft. In some embodiments a cell-reactivecompstatin analog is administered systemically, e.g., intravenously.

The invention provides a composition comprising: (a) an isolated graft;and (b) a cell-reactive, long-acting, or targeted compstatin analog. Insome embodiments the composition further comprises a liquid solutionsuitable for contacting (e.g., suitable for rinsing, washing, bathing,perfusing, maintaining, or storing) a graft (e.g., an organ) such as anisolated graft that has been removed from a donor and is awaitingtransplantation to a recipient. In some embodiments the inventionprovides a composition comprising: (a) a liquid solution suitable forcontacting a graft (e.g., an organ); and (b) a cell-reactive,long-acting, or targeted compstatin analog. The liquid solution can beany liquid solution that is physiologically acceptable to the graft(e.g., appropriate osmotic composition, non-cytotoxic) and medicallyacceptable in view of the subsequent introduction of the graft into therecipient (e.g., preferably sterile or at least reasonably free frommicroorganisms or other contaminants) and compatible with thecell-reactive compstatin analog (i.e., will not destroy the reactivityof the compstatin analog) or compatible with the long-acting or targetedcompstatin analog. In some embodiments, a solution is any solution ownin the art for any such purposes. In some embodiments, a liquid solutionis Marshall's or Hyperosmolar Citrate (Soltran®, Baxter Healthcare),University of Wisconsin (UW) solution (ViaSpan™, Bristol Myers Squibb),Histidine Tryptophan Ketoglutarate (HTK) solution (Custodial®, KohlerMedical Limited), EuroCollins (Fresenius), and Celsior® (SangstatMedical), Polysol, IGL-1, or AQIX® RS-1. Of course other solutions,e.g., containing equivalent or similar ingredients in the same ordifferent concentrations could be used within the scope ofphysiologically acceptable compositions. In some embodiments a solutiondoes not contain ingredient(s) with which the cell-reactive compstatinanalog would be expected to significantly react, and any solution may bemodified or designed to lack such ingredients. In some embodiments, thecell-reactive compstatin analog is present in the graft-compatiblesolution at a concentration of, e.g., between 0.01 mg/ml and 100 mg/mlor may be added to the solution to achieve such concentration.

In some embodiments, the invention provides a kit comprising: (a) acell-reactive, long-acting, or targeted compstatin analog; and (b) agraft-compatible solution or solid (e.g., powder) components thereof.The cell-reactive, long-acting, or targeted compstatin analog may beprovided in solid form (e.g., powder) or at least in part dissolved in asolution. In some embodiments the cell-reactive, long-acting, ortargeted compstatin analog and/or graft-compatible solution are providedin predetermined amounts, so that when combined, a solution ofappropriate concentration for contacting a graft with the cell-reactive,long-acting, or targeted compstatin analog is produced. In manyembodiments the cell-reactive, long-acting, or targeted compstatinanalog and graft-compatible solution or solid (e.g., powder) componentsthereof are in separate containers within the kit. In some embodimentsthe cell-reactive compstatin analog and components of a graft-compatiblesolution are both provided in solid (e.g., powder) form, either inseparate containers or mixed. In some embodiments the kit comprisesinstructions for use, e.g., instructions for adding a cell-reactive,long-acting, or targeted compstatin analog to a graft-compatiblesolution and/or instructions for contacting a graft with a cell-reactivecompstatin analog. Optionally the kit contains a label approved by agovernment agency responsible for regulating products used intransplantation, cell therapy, and/or blood transfusion.

The invention further provides a method of covalently attaching acompstatin analog to an isolated graft comprising contacting theisolated graft with a cell-reactive compstatin analog. The inventionfurther provides an isolated graft having a compstatin analog covalentlyattached thereto. Typically the isolated graft has many molecules ofcompstatin analog attached thereto. In some embodiments, a graft is orcomprises a solid organ such as a kidney, liver, lung, pancreas, orheart. In some embodiments, a graft is or comprises bone, cartilage,fascia, tendon, ligament, cornea, sclera, pericardium, skin, heartvalve, blood vessel, amniotic membrane, or dura mater. In someembodiments, a graft comprises multiple organs such as a heart-lung orpancreas-kidney graft. In some embodiments, a graft comprises less thana complete organ or tissue. For example, a graft may contain a portionof an organ or tissue, e.g., a liver lobe, section of blood vessel, skinflap, or heart valve. In some embodiments, a graft comprises apreparation comprising isolated cells or tissue fragments that have beenisolated from their tissue of origin but retain at least some tissuearchitecture, e.g., pancreatic islets. In some embodiments, apreparation comprises isolated cells that are not attached to each othervia connective tissue, e.g., hematopoietic stem cells or progenitorcells derived from peripheral and/or cord blood, or whole blood or anycell-containing blood product such as red blood cells (RBCs) orplatelets. In some embodiments a graft is obtained from a deceased donor(e.g., a “donation after brain death” (DBD) donor or “donation aftercardiac death” donor). In some embodiments, depending on the particulartype of graft, a graft is obtained from a living donor. For example,kidneys, liver sections, blood cells, are among the types of grafts thatcan often be obtained from a living donor without undue risk to thedonor and consistent with sound medical practice.

In some embodiments, a graft is a xenograft (i.e., the donor andrecipient are of different species). In some embodiments a graft is anautograft (i.e., a graft from one part of the body to another part ofthe body in the same individual). In some embodiments, a graft is anisograft (i.e., the donor and recipient are genetically identical). Inmost embodiments, the graft is an allograft (i.e., the donor andrecipient are genetically non-identical members of the same species). Inthe case of an allograft, the donor and recipient may or may not begenetically related (e.g., family members). Typically, the donor andrecipient have compatible blood groups (at least ABO compatibility andoptionally Rh, Kell and/or other blood cell antigen compatibility). Therecipient's blood may have been screened for alloantibodies to the graftand/or the recipient and donor since the presence of such antibodies canlead to hyperacute rejection (i.e., rejection beginning almostimmediately, e.g., within several minutes after the graft comes intocontact with the recipient's blood). A complement-dependent cytotoxicity(CDC) assay can be used to screen a subject's serum for anti-HLAantibodies. The serum is incubated with a panel of lymphocytes of knownHLA phenotype. If the serum contains antibodies against HLA molecules onthe target cells, cell death due to complement-mediated lysis occurs.Using a selected panel of target cells allows one to assign specificityto the detected antibody. Other techniques useful for determining thepresence or absence anti-HLA antibodies and, optionally, determiningtheir HLA specificity, include ELISA assays, flow cytometry assays,microbead array technology (e.g., Luminex technology). The methodologyfor performing these assays is well known, and a variety of kits forperforming them are commercially available.

In some embodiments a cell-reactive, long-acting, or targeted compstatinanalog inhibits complement-mediated rejection. For example, in someembodiments a cell-reactive, long-acting, or targeted compstatin analoginhibits hyperacute rejection. Hyperacute rejection is caused at leastin part by antibody-mediated activation of the recipient's complementsystem via the classical pathway and resulting MAC deposition on thegraft. It typically results from the presence in the recipient ofpre-existing antibodies that react with the graft. While it is desirableto attempt to avoid hyperacute rejection by appropriate matching priorto transplantation, it may not always possible to do so due, e.g., totime and/or resource constraints. Furthermore, some recipients (e.g.,multiply transfused individuals, individuals who have previouslyreceived transplants, women who have had multiple pregnancies) mayalready have so many pre-formed antibodies, potentially includingantibodies to antigens that are not typically tested for, that it can bedifficult or perhaps almost impossible to obtain with confidence acompatible graft in a timely manner. Such individuals are at increasedrisk of hyperacute rejection.

In some embodiments, a cell-reactive, long-acting, or targetedcompstatin analog inhibits acute rejection or graft failure. As usedherein, “acute rejection” refers to rejection occurring between at least24 hours, typically at least several days to a week, after a transplant,up to 6 months after the transplant. Acute antibody-mediated rejection(AMR) often involves an acute rise in donor-specific alloantibody (DSA)in the first few weeks after transplantation. Without wishing to bebound by any theory, it is possible that pre-existing plasma cellsand/or the conversion of memory B cells to new plasma cells play a rolein the increased DSA production. Such antibodies can result incomplement-mediated damage to the graft, which can be inhibited bycontacting the graft with a cell-reactive compstatin analog. Withoutwishing to be bound by any theory, inhibiting complement activation atthe graft may reduce leukocyte (e.g., neutrophil) infiltration, anothercontributor to acute graft failure.

In some embodiments, a cell-reactive, long-acting, or targetedcompstatin analog inhibits complement-mediated I/R injury to a graft. Asdiscussed further below, I/R injury can occur upon reperfusion of tissuewhose blood supply has been temporarily disrupted, as occurs intransplanted organs. Reducing I/R injury would reduce the likelihood ofacute graft dysfunction or reduce its severity, and reduce thelikelihood of acute graft failure.

In some embodiments, a cell-reactive, long-acting, or targetedcompstatin analog inhibits chronic rejection and/or chronic graftfailure. As used herein, “chronic rejection or graft failure” refers torejection or failure occurring at least 6 months post-transplant, e.g.,between 6 months and 1, 2, 3, 4, 5 years, or more post-transplant, oftenafter months to years of good graft function. It is caused by a chronicinflammatory and immune response against the graft. For purposes hereof,chronic rejection can include chronic allograft vasculopathy, a termused to refer to fibrosis of the internal blood vessels of thetransplanted tissue. As immunosuppressive regimens have reduced theincidence of acute rejection, chronic rejection is becoming moreprominent as a cause of graft dysfunction and failure. There isincreasing evidence that B-cell production of alloantibody is animportant element in the genesis of chronic rejection and graft failure(Kwun J. and Knechtle S J, Transplantation, 88(8):955-61 (2009). Earlierdamage to the graft may be a contributing factor leading to chronicprocesses such as fibrosis that can ultimately lead to chronicrejection. Thus, inhibiting such earlier damage using a cell-reactivecompstatin analog may delay and/or reduce the likelihood or severity ofchronic graft rejection.

In some embodiments, a long-acting compstatin analog is administered toa graft recipient to inhibit graft rejection and/or graft failure.

C. Ischemia/Reperfusion Injury

Ischemia-reperfusion (I/R) injury is an important cause of tissue damagefollowing trauma and in other conditions associated with temporarydisruption of blood flow such as myocardial infarction, stroke, severeinfection, vascular disease, aneurysm repair, cardiopulmonary bypass,and transplantation.

In the setting of trauma, systemic hypoxemia, hypotension, and localinterruption of the blood supply resulting from contusions, compartmentsyndrome, and vascular injuries cause ischemia that damagesmetabolically active tissues. Restoration of the blood supply triggersan intense systemic inflammatory reaction that is often more harmfulthan the ischemia itself. Once the ischemic region is reperfused,factors that are produced and released locally enter the circulatorysystem and reach remote locations, sometimes causing significant damageto organs not affected by the original ischemic insult, such as thelungs and intestine, leading to single and multiple organ dysfunction.Complement activation occurs soon after reperfusion and is a keymediator of post-ischemic damage, both directly and through itschemoattractive and stimulatory effects on neutrophils. All three majorcomplement pathways are activated and, acting cooperatively orindependently, are involved in I/R related adverse events affectingnumerous organ systems. In some embodiments of the invention, acell-reactive, long-acting, or targeted compstatin analog isadministered to a subject who has recently (e.g., within the preceding2, 4, 8, 12, 24, or 48 hours) experienced trauma, e.g., trauma that putsthe subject at risk of I/R injury, e.g., due to systemic hypoxemia,hypotension, and/or local interruption of the blood supply. In someembodiments the cell-reactive compstatin analog may be administeredintravascularly, optionally into a blood vessel that supplies an injuredbody part or directly to the body part. In some embodiments, the subjectsuffers from spinal cord injury, traumatic brain injury, burn, and/orhemorrhagic shock.

In some embodiments, a cell-reactive, long-acting, or targetedcompstatin analog is administered to a subject prior to, during, orafter a surgical procedure, e.g., a surgical procedure that is expectedto temporarily disrupt blood flow to a tissue, organ, or portion of thebody. Examples of such procedures include cardiopulmonary bypass,angioplasty, heart valve repair/replacement, aneurysm repair, or othervascular surgeries. The cell-reactive compstatin analog may beadministered prior to, after, and/or during an overlapping time periodwith the surgical procedure.

In some embodiments, a cell-reactive, long-acting, or targetedcompstatin analog is administered to a subject who has suffered an MI,thromboembolic stroke, deep vein thrombosis, or pulmonary embolism. Thecell-reactive compstatin analog may be administered in combination witha thrombolytic agent such as tissue plasminogen activator (tPA) (e.g.,alteplase (Activase), reteplase (Retavase), tenecteplase (TNKase)),anistreplase (Eminase), streptokinase (Kabikinase, Streptase), orurokinase (Abbokinase). The cell-reactive, long-acting, or targetedcompstatin analog may be administered prior to, after, and/or during anoverlapping time period with the thrombolytic agent.

In some embodiments, a cell-reactive, long-acting, or targetedcompstatin analog is administered to a subject to treat FR injury.

D. Other Complement-Mediated Disorders

In some embodiments, a cell-reactive, long-acting, or targetedcompstatin analog is introduced into the eye for treatment of an eyedisorder such as age-related macular degeneration (AMD), diabeticretinopathy, glaucoma, or uveitis. For example, a cell-reactivecompstatin analog may be introduced into the vitreous cavity (e.g., byintravitreal injection), for treatment of a subject at suffering from orat risk of AMD. In some embodiments a cell-reactive, long-acting, ortargeted compstatin analog is introduced into the anterior chamber,e.g., to treat anterior uveitis.

In some embodiments a cell-reactive, long-acting, or targeted compstatinanalog is used to treat a subject suffering from or at risk of anautoimmune disease, e.g., an autoimmune disease mediated at least inpart by antibodies against one or more self antigens.

Cell-reactive, long-acting, or targeted compstatin analogs may beintroduced into the synovial cavity, e.g., in a subject suffering fromarthritis (e.g., rheumatoid arthritis). Of course they may beadministered systemically in addition or

In some embodiments, a cell-reactive, long-acting, or targetedcompstatin analog is used to treat a subject suffering from or at riskof an intracerebral hemorrhage.

In some embodiments a cell-reactive, long-acting, or targeted compstatinanalog is used to treat a subject suffering from or at risk ofmyasthenia gravis.

In some embodiments a cell-reactive, long-acting, or targeted compstatinanalog is used to treat a subject suffering from or at risk ofneuromyelitis optica (NMO),

In some embodiments a cell-reactive, long-acting, or targeted compstatinanalog is used to treat a subject suffering from or at risk ofmembranoproliferative glomerulitis (MPGN), e.g., MPGN type I, MPGN typeII, or MPGH type III.

In some embodiments a cell-reactive, long-acting, or targeted compstatinanalog is used to treat a subject suffering from or at risk of aneurodegenerative disease. In some embodiments a cell-reactive,long-acting, or targeted compstatin analog is used to treat a subjectsuffering from neuropathic pain or at risk of developing neuropathicpain. In some embodiments a cell-reactive, long-acting, or targetedcompstatin analog is used to treat a subject suffering from or at riskof rhinosinusitis or nasal polyposis. In some embodiments acell-reactive, long-acting, or targeted compstatin analog is used totreat a subject suffering from or at risk of cancer. In some embodimentsa cell-reactive, long-acting, or targeted compstatin analog is used totreat a subject suffering from or at risk of sepsis. In some embodimentsa cell-reactive, long-acting, or targeted compstatin analog is used totreat a subject suffering from or at risk of adult respiratory distresssyndrome.

In some embodiments a cell-reactive, long-acting, or targeted compstatinanalog is used to treat a subject suffering from or at risk ofanaphylaxis or infusion reaction. For example, in some embodiments asubject may be pretreated prior to, during, or after receiving a drug ora vehicle that may cause anaphylaxis or infusion reaction. In someembodiments a subject at risk of or suffering from anaphylaxis from afood (e.g., peanut, shellfish, or other food allergens), insect sting(e.g., bee, wasp), is treated with a cell-reactive, long-acting, ortargeted compstatin analog.

The cell-reactive long-acting, or targeted compstatin analog may beadministered locally or systemically, in various embodiments of theinvention.

In some embodiments, a cell-reactive, long-acting, or targetedcompstatin analog is used to treat a respiratory disease, e.g., asthmaor chronic obstructive pulmonary disease (COPD). The cell-reactive,long-acting, or targeted compstatin analog may, for example, beadministered to the respiratory tract by inhalation, e.g., as a drypowder or via nebulization, or may be administered by injection, e.g.,intravenously, intramuscularly, or subcutenously, in variousembodiments. In some embodiments, a cell-reactive, long-acting, ortargeted compstatin analog is used to treat severe asthma, e.g., asthmathat is not sufficiently controlled by bronchodilators and/or inhaledcorticosteroids.

In some aspects, methods of treating a complement-mediated disorder,e.g., a chronic complement-mediated disorder, are provided, the methodscomprising administering a long-acting complement inhibitor to a subjectin need of treatment for the disorder. The long-acting compstatin analogmay be any long-acting compstatin analog described herein, in variousembodiments. In some aspects, methods of treating a Th17-associateddisorder are provided, the methods comprising administering along-acting complement inhibitor to a subject in need of treatment forthe disorder.

In some aspects, a “chronic disorder” is a disorder that persists for atleast 3 months and/or is accepted in the art as being a chronicdisorder. In many embodiments, a chronic disorder persists for at least6 months, e.g., at least 1 year, or more, e.g., indefinitely. One ofordinary skill in the art will appreciate that at least somemanifestations of various chronic disorders may be intermittent and/ormay wax and wane in severity over time. A chronic disorder may beprogressive, e.g., having a tendency to become more severe or affectlarger areas over time. A number of chronic complement-mediateddisorders are discussed herein. A chronic complement-mediated disordermay be any chronic disorder in which complement activation (e.g.,excessive or inappropriate complement activation) is involved, e.g., asa contributing and/or at least partially causative factor. Forconvenience, disorders are sometimes grouped by reference to an organ orsystem that is often particularly affected in subjects suffering fromthe disorder. It will be appreciated that a number of disorders canaffect multiple organs or systems, and such classification(s) are in noway limiting. Furthermore, a number of manifestations (e.g., symptoms)may occur in subjects suffering from any of a number of differentdisorders. Non-limiting information regarding disorders of interestherein may be found, e.g., in standard textbooks of internal medicinesuch as Cecil Textbook of Medicine (e.g., 23rd edition), Harrison'sPrinciples of Internal Medicine (e.g., 17th edition), and/or standardtextbooks focusing on particular areas of medicine, particular bodysystems or organs, and/or particular disorders.

In some embodiments, a chronic complement-mediated disorder is aTh2-associated disorder. As used herein, a Th2-associated disorder is adisorder characterized by an excessive number and/or excessive orinappropriate activity of CD4+ helper T cells of the Th2 subtype (“Th2cells”) in the body or a portion thereof, e.g., in at least one tissue,organ, or structure. For example, there may be a predominance of Th2cells relative to CD4+ helper T cells of the Th1 subtype (“Th1 cells”)e.g., in at least one tissue, organ, or structure affected by adisorder. As known in the art, Th2 cells typically secretecharacteristic cytokines such as interleukin-4 (IL-4), interleukin-5(IL-5), and interleukin-13 (IL-13), while Th1 cells typically secreteinterferon-γ (IFN-γ) and tumor necrosis factor β (TNF β). In someembodiments, a Th2-associated disorder is characterized by excessiveproduction and/or amount of IL-4, IL-5, and/or IL-13, e.g., relative toIFN-γ and/or TNF β e.g., in at least some at least one tissue, organ, orstructure

In some embodiments, a chronic complement-mediated disorder is aTh17-associated disorder. In some aspects, as described in furtherdetail in PCT/US2012/043845, filed Jun. 22, 2012, entitled “Methods ofTreating Chronic Disorders with Complement Inhibitors”, complementactivation and Th17 cells participate in a cycle that involves dendriticcells and antibodies and that contributes to maintenance of a pathologicimmunologic microenvironment underlying a range of disorders. Withoutwishing to be bound by any theory, the pathologic immunologicmicroenvironment, once established, is self-sustaining and contributesto cell and tissue injury. In some aspects, long-acting compstatinanalogs are of use to treat Th17-associated disorders.

As used herein, a Th17-associated disorder is a disorder characterizedby an excessive number and/or excessive or inappropriate activity ofCD4+ helper T cells of the Th17 subtype (“Th17 cells”) in the body or aportion thereof, e.g., in at least one tissue, organ, or structure. Forexample, there may be a predominance of Th17 cells relative to Th1and/or Th2 cells, e.g., in at least one tissue, organ, or structureaffected by a disorder. In some embodiments a predominance of Th17 cellsis a relative predominance, e.g., the ratio of Th17 cells to Th1 cellsand/or the ratio of Th17 cells to Th2 cells, is increased relative tonormal values. In some embodiments the ratio of Th17 cells to Tregulatory cells (CD4⁺CD25⁺ regulatory T cells, also termed “Tregcells”), is increased relative to normal values. Formation of Th17 cellsand/or activation of Th17 cells is promoted by various cytokines, e.g.,interleukin 6 (IL-6), interleukin 21 (IL-21), interleukin 23 (IL-23),and/or interleukin 1β (IL-1β). Formation of Th17 cells encompassesdifferentiation of precursor T cells, e.g., naïve CD4+ T cells, towardsa Th17 phenotype and their maturation into functional Th17 cells. Insome embodiments, formation of Th17 cells encompasses any aspect ofdevelopment, proliferation (expansion), survival, and/or maturation ofTh17 cells. In some embodiments, a Th17-associated disorder ischaracterized by excessive production and/or amount of IL-6, IL-21,IL-23, and/or IL-1β. Th17 cells typically secrete characteristiccytokines such as interleukin-17A (IL-17A), interleukin-17F (IL-17F),interleukin-21 (IL-21), and interleukin-22 (IL-22). In some embodiments,a Th17-associated disorder is characterized by excessive productionand/or amount of a Th17 effector cytokine, e.g., IL-17A, IL-17F, IL-21,and/or IL-22. In some embodiments excessive production or amount of acytokine is detectable in the blood. In some embodiments excessiveproduction or amount of a cytokine is detectable locally, e.g., in atleast one tissue, organ or structure. In some embodiments aTh17-associated disorder is associated with a decreased number of Tregsand/or decreased amount of a Treg-associated cytokine. In someembodiments a Th17 disorder is any chronic inflammatory disease, whichterm encompasses a range of ailments characterized by self-perpetuatingimmune insults to a variety of tissues and that seem to be dissociatedfrom the initial insult that caused the ailment (which may be unknown).In some embodiments a Th17-associated disorder is any autoimmunedisease. Many if not most “chronic inflammatory diseases” may in fact beauto-immune diseases. Examples of Th17-associated disorders includeinflammatory skin diseases such as psoriasis and atopic dermatitis;systemic scleroderma and sclerosis; inflammatory bowel disease (IBD)(such as Crohn's disease and ulcerative colitis); Behcet's Disease;dermatomyositis; polymyositis; multiple sclerosis (MS); dermatitis;meningitis; encephalitis; uveitis; osteoarthritis; lupus nephritis;rheumatoid arthritis (RA), Sjogren's syndrome, multiple sclerosis,vasculitis; central nervous system (CNS) inflammatory disorders, chronichepatitis; chronic pancreatitis, glomerulonephritis; sarcoidosis;thyroiditis, pathologic immune responses to tissue/organ transplantation(e.g., transplant rejection); COPD, asthma, bronchiolitis,hypersensitivity pneumonitis, idiopathic pulmonary fibrosis (IPF),periodontitis, and gingivitis. In some embodiments a Th17 disease is aclassically known auto-immune disease such as Type I diabetes orpsoriasis. In some embodiments a Th17-associated disorder is age-relatedmacular degeneration.

In some embodiments, a chronic complement-mediated disorder is anIgE-associated disorder. As used herein, an “IgE-associated disorder” isa disorder characterized by excessive and/or inappropriate productionand/or amount of IgE, excessive or inappropriate activity of IgEproducing cells (e.g., IgE producing B cells or plasma cells), and/orexcessive and/or inappropriate activity of IgE responsive cells such aseosinophils or mast cells. In some embodiments, an IgE-associateddisorder is characterized by elevated levels of total IgE and/or in someembodiments, allergen-specific IgE, in the plasma of a subject and/orlocally.

In some embodiments, a chronic complement-mediated disorder ischaracterized by the presence of autoantibodies and/or immune complexesin the body, which may activate complement via, e.g., the classicalpathway. Autoantibodies may, for example, bind to self antigens, e.g.,on cells or tissues in the body. In some embodiments, autoantibodiesbind to antigens in blood vessels, skin, nerves, muscle, connectivetissue, heart, kidney, thyroid, etc. In some embodiments, a chroniccomplement-mediated disorder is not characterized by autoantibodiesand/or immune complexes.

In some embodiments, a chronic complement-mediated disorder is arespiratory disorder. In some embodiments, a chronic respiratorydisorder is asthma or chronic obstructive pulmonary disease (COPD). Insome embodiments, a chronic respiratory disorder is pulmonary fibrosis(e.g., idiopathic pulmonary fibrosis), radiation-induced lung injury,allergic bronchopulmonary aspergillosis, hypersensitivity pneumonitis(also known as allergic alveolitis), eosinophilic pneumonia,interstitial pneumonia, sarcoid, Wegener's granulomatosis, orbronchiolitis obliterans. In some embodiments, the invention provides amethod of treating a subject in need of treatment for a chronicrespiratory disorder, e.g., asthma, COPD, pulmonary fibrosis,radiation-induced lung injury, allergic bronchopulmonary aspergillosis,hypersensitivity pneumonitis (also known as allergic alveolitis),eosinophilic pneumonia, interstitial pneumonia, sarcoid, Wegener'sgranulomatosis, or bronchiolitis obliterans, the method comprisingadministering a long-acting complement inhibitor to a subject in need oftreatment for the disorder.

In some embodiments, a chronic complement-mediated disorder is allergicrhinitis, rhinosinusitis, or nasal polyposis. In some embodiments, theinvention provides a method of treating a subject in need of treatmentfor allergic rhinitis, rhinosinusitis, or nasal polyposis, the methodcomprising administering a long-acting complement inhibitor to a subjectin need of treatment for the disorder.

In some embodiments, a chronic complement-mediated disorder is adisorder that affects the musculoskeletal system. Examples of suchdisorders include inflammatory joint conditions (e.g., arthritis such asrheumatoid arthritis or psoriatic arthritis, juvenile chronic arthritis,spondyloarthropathies Reiter's syndrome, gout). In some embodiments, amusculoskeletal system disorder results in symptoms such as pain,stiffness and/or limitation of motion of the affected body part(s).Inflammatory myopathies include dermatomyositis, polymyositis, andvarious others are disorders of chronic muscle inflammation of unknownetiology that result in muscle weakness. In some embodiments, a chroniccomplement-mediated disorder is myasthenia gravis. In some embodiments,the invention provides a method of treating any of the foregoingdisorders affecting the musculoskeletal system, the method comprisingadministering a long-acting complement inhibitor to a subject in need oftreatment for the disorder.

In some embodiments, a chronic complement-mediated disorder is adisorder that affects the integumentary system. Examples of suchdisorders include, e.g., atopic dermatitis, psoriasis, pemphigus,systemic lupus erythematosus, dermatomyositis, scleroderma,sclerodermatomyositis, Sjögren syndrome, and chronic urticaria. In someaspects, the invention provides a method of treating any of theforegoing disorders affecting the integumentary system, the methodcomprising administering a long-acting complement inhibitor to a subjectin need of treatment for the disorder.

In some embodiments, a chronic complement-mediated disorder affects thenervous system, e.g., the central nervous system (CNS) and/or peripheralnervous system (PNS). Examples of such disorders include, e.g., multiplesclerosis, other chronic demyelinating diseases, amyotrophic lateralsclerosis, chronic pain, stroke, allergic neuritis, Huntington'sdisease, Alzheimer's disease, and Parkinson's disease. In someembodiments, the invention provides a method of treating any of theforegoing disorders affecting the nervous system, the method comprisingadministering a complement inhibitor according to a dosing scheduledescribed herein to a subject in need of treatment for the disorder.

In some embodiments, a chronic complement-mediated disorder affects thecirculatory system. For example, in some embodiments the disorder is avasculitis or other disorder associated with vessel inflammation, e.g.,blood vessel and/or lymph vessel inflammation. In some embodiments, avasculitis is polyarteritis nodosa, Wegener's granulomatosis, giant cellarteritis, Churg-Strauss syndrome, microscopic polyangiitis,Henoch-Schonlein purpura, Takayasu's arteritis, Kawasaki disease, orBehcet's disease. In some embodiments, a subject, e.g., a subject inneed of treatment for vasculitis, is positive for antineutrophilcytoplasmic antibody (ANCA).

In some embodiments, a chronic complement-mediated disorder affects thegastrointestinal system. For example, the disorder may be inflammatorybowel disease, e.g., Crohn's disease or ulcerative colitis. In someembodiments, the invention provides a method of treating a chroniccomplement-mediated disorder that affects the gastrointestinal system,the method comprising administering a long-acting complement inhibitorto a subject in need of treatment for the disorder.

In some embodiments, a chronic complement-mediated disorder is athyroiditis (e.g., Hashimoto's thryoiditis, Graves' disease, post-partumthryoiditis), myocarditis, hepatitis (e.g., hepatitis C), pancreatitis,glomerulonephritis (e.g., membranoproliferative glomerulonephritis ormembranous glomerulonephritis), or panniculitis.

In some embodiments, the invention provides methods of treating asubject suffering from chronic pain, the methods comprisingadministering a long-acting complement inhibitor to a subject in needthereof. In some embodiments, a subject suffers from neuropathic pain.Neuropathic pain has been defined as pain initiated or caused by aprimary lesion or dysfunction in the nervous system, in particular, painarising as a direct consequence of a lesion or disease affecting thesomatosensory system. For example, neuropathic pain may arise fromlesions that involve the somatosensory pathways with damage to smallfibres in peripheral nerves and/or to the spino-thalamocortical systemin the CNS. In some embodiments, neuropathic pain arises from autoimmunedisease (e.g., multiple sclerosis), metabolic disease (e.g., diabetes),infection (e.g., viral disease such as shingles or HIV), vasculardisease (e.g., stroke), trauma (e.g., injury, surgery), or cancer. Forexample, neuropathic pain can be pain that persists after healing of aninjury or after cessation of a stimulus of peripheral nerve endings orpain that arises due to damage to nerves. Exemplary conditions of orassociated with neuropathic pain include painful diabetic neuropathy,post-herpetic neuralgia (e.g., pain persisting or recurring at the siteof acute herpes zoster 3 or more months after the acute episode),trigeminal neuralgia, cancer related neuropathic pain,chemotherapy-associated neuropathic pain, HIV-related neuropathic pain(e.g., from HIV neuropathy), central/post-stroke neuropathic pain,neuropathy associated with back pain, e.g., low back pain (e.g., fromradiculopathy such as spinal root compression, e.g., lumbar rootcompression, which compression may arise due to disc herniation), spinalstenosis, peripheral nerve injury pain, phantom limb pain,polyneuropathy, spinal cord injury related pain, myelopathy, andmultiple sclerosis. In certain embodiments of the invention a complementinhibitor is administered according to an inventive dosing schedule totreat neuropathic pain in a subject with one or more of theafore-mentioned conditions.

In some embodiments, a chronic complement-mediated disorder is a chroniceye disorder. In some embodiments, the chronic eye disorder ischaracterized by macular degeneration, choroidal neovascularization(CNV), retinal neovascularization (RNV), ocular inflammation, or anycombination of the foregoing. Macular degeneration, CNV, RNV, and/orocular inflammation may be a defining and/or diagnostic feature of thedisorder. Exemplary disorders that are characterized by one or more ofthese features include, but are not limited to, macular degenerationrelated conditions, diabetic retinopathy, retinopathy of prematurity,proliferative vitreoretinopathy, uveitis, keratitis, conjunctivitis, andscleritis. Macular degeneration related conditions include, e.g.,age-related macular degeneration (AMD). In some embodiments, a subjectis in need of treatment for wet AMD. In some embodiments, a subject isin need of treatment for dry AMD. In some embodiments, a subject is inneed of treatment for geographic atrophy (GA). In some embodiments, asubject is in need of treatment for ocular inflammation. Ocularinflammation can affect a large number of eye structures such as theconjunctiva (conjunctivitis), cornea (keratitis), episclera, sclera(scleritis), uveal tract, retina, vasculature, and/or optic nerve.Evidence of ocular inflammation can include the presence ofinflammation-associated cells such as white blood cells (e.g.,neutrophils, macrophages) in the eye, the presence of endogenousinflammatory mediator(s), one or more symptoms such as eye pain,redness, light sensitivity, blurred vision and floaters, etc. Uveitis isa general term that refers to inflammation in the uvea of the eye, e.g.,in any of the structures of the uvea, including the iris, ciliary bodyor choroid. Specific types of uveitis include iritis, iridocyclitis,cyclitis, pars planitis and choroiditis. In some embodiments, a subjectis in need of treatment for geographic atrophy (GA). In someembodiments, the chronic eye disorder is an eye disorder characterizedby optic nerve damage (e.g., optic nerve degeneration), such asglaucoma.

As noted above, in some embodiments the chronic respiratory disease isasthma. Information regarding risk factors, epidemiology, pathogenesis,diagnosis, current management of asthma, etc., may be found, e.g., in“Expert Panel Report 3: Guidelines for the Diagnosis and Management ofAsthma”. National Heart Lung and Blood Institute. 2007.http://www.nhlbi.nih.gov/guidelines/asthma/asthgdln.pdf. (“NHLBIGuidelines”; www.nhlbi.nih.gov/guidelines/asthma/asthgdln.htm), GlobalInitiative for Asthma, Global Strategy for Asthma Management andPrevention 2010 “GINA Report”) and/or standard textbooks of internalmedicine such as Cecil Textbook of Medicine (20th edition), Harrison'sPrinciples of Internal Medicine (17th edition), and/or standardtextbooks focusing on pulmonary medicine. Asthma is a chronicinflammatory disorder of the airways in which many cells and cellularelements play a role, such as, mast cells, eosinophils, T lymphocytes,macrophages, neutrophils, and epithelial cells Asthmatic individualsexperience recurrent episodes associated with symptoms such as wheezing,breathlessness (also termed dyspnea or shortness of breath), chesttightness, and coughing. These episodes are usually associated withwidespread but variable airflow obstruction that is often reversible,either spontaneously or with treatment. The inflammation also causes anassociated increase in the existing bronchial hyperresponsiveness to avariety of stimuli. Airway hyperresponsiveness (an exaggeratedbronchoconstrictor response to stimuli) is a typical feature of asthma.In general, airflow limitation results from bronchoconstriction andairway edema. Reversibility of airflow limitation may be incomplete insome patients with asthma. For example, airway remodeling can lead tofixed airway narrowing. Structural changes can include thickening of thesub-basement membrane, subepithelial fibrosis, airway smooth musclehypertrophy and hyperplasia, blood vessel proliferation and dilation,and mucous gland hyperplasia, and hypersecretion.

Individuals with asthma may experience exacerbations, which areidentified as events characterized by a change from the individual'sprevious status. Severe asthma exacerbations can be defined as eventsthat require urgent action on the part of the individual and his/herphysician to prevent a serious outcome, such as hospitalization or deathfrom asthma. For example, a severe asthma exacerbation may require useof systemic corticosteroids (e.g., oral corticosteroids) in a subjectwhose asthma is usually well controlled without OCS or may require anincrease in a stable maintenance dose. Moderate asthma exacerbations canbe defined as events that are troublesome to the subject, and thatprompt a need for a change in treatment, but that are not severe. Theseevents are clinically identified by being outside the subject's usualrange of day-to-day asthma variation.

Current medications for asthma are typically categorized into twogeneral classes: long-term control medications (“controllermedications”) such as inhaled corticosteroids (ICS), oralcorticosteroids (OCS), long-acting bronchodilators (LABAs), leukotrienemodifiers (e.g., leukotriene receptor antagonists or leukotrienesynthesis inhibitors, anti-IgE antibodies (omalizumab (Xolair®)),cromolyn and nedocromil, which are used to achieve and maintain controlof persistent asthma and quick-relief medications such as short-actingbronchodilators (SABAs), which are used to treat acute symptoms andexacerbations. For purposes of the present invention, these treatmentsmay be referred to as “conventional therapy”. Treatment of exacerbationsmay also include increasing the dose and/or intensity of controllermedication therapy. For example, a course of OCS can be used to regainasthma control. Current guidelines mandate daily administration ofcontroller medication or, in many cases, administration of multipledoses of controller medication each day for subjects with persistentasthma (with the exception of Xolair, which is administered every 2 or 4weeks).

A subject is generally considered to have persistent asthma if thesubject suffers from symptoms on average more than twice a week and/ortypically uses a quick relief medication (e.g., SABA) more than twice aweek for symptom control. “Asthma severity” can be classified based onthe intensity of treatment required to control the subject's asthma oncerelevant comorbidities have been treated and inhaler technique andadherence have been optimized (see, e.g., GINA Report; Taylor, DR, EurRespir J 2008; 32:545-554). The description of treatment intensity canbe based on the medications and doses recommended in the stepwisetreatment algorithm found in guidelines such as NHLBI Guidelines 2007,GINA Report, and their predecessors and/or in standard medicaltextbooks. For example, asthma can be classified as intermittent, mild,moderate, or severe as indicated in Table 2, where “treatment” refers totreatment sufficient to achieve subject's best level of asthma control.(It will be understood that the categories of mild, moderate, and severeasthma in general imply persistent rather than intermittent asthma). Oneof ordinary skill in the art will appreciate that Table 2 is exemplary,and that not all of these medications will be available in allhealthcare systems, which may affect the assessment of asthma severityin some environments. It will also be appreciated that other emerging ornew approaches may affect the classification of mild/moderate asthma.However, the same principle, of mild asthma being defined by the abilityto achieve good control using very low-intensity treatment and severeasthma being defined by the requirement for high-intensity treatment,can still be applied. Asthma severity can also or alternately beclassified based on intrinsic intensity of the disease in the absence oftreatment (see, e.g., NHBLI Guidelines 2007). Assessment can be made onthe basis of current spirometry and the patient's recall of symptomsover the previous 2-4 weeks. Parameters of current impairment and futurerisk may be assessed and included in a determination of the level ofasthma severity. In some embodiments, asthma severity is defined asshown in FIG. 3.4(a), 3.4(b), 3.4(c) of the NHBLI Guidelines, forindividuals 0-4, 5-11, or ≥12 years of age, respectively.

TABLE 2 Treatment-based Asthma Classification Asthma ClassificationTreatment Intermittent SABA as needed (typically no more than twice aweek) Mild Low-dose ICS or other low-intensity treatment (e.g., LTRA,cromolyn, nedocromil, theophylline) Moderate Low to moderate dose ICSand LABA or other extra treatment Severe High-intensity treatment(high-dose ICS and LABA ± oral corticosteroids and/or other extratreatment)

“Asthma control” refers to the extent to which the manifestations ofasthma have been reduced or removed by treatment (whetherpharmacological or non-pharmacological). Asthma control can be assessedbased on factors such as symptom frequency, nighttime symptoms,objective measures of lung function such as spirometry parameters (e.g.,% FEV₁ of predicted, FEV₁ variability, requirement for use of SABA forsymptom control. Parameters of current impairment and future risk may beassessed and included in a determination of the level of asthma control.In some embodiments, asthma control is defined as shown in FIG. 4.3(a),4.3(b), or 4.3(c) of NHBLI Guidelines, for individuals 0-4, 5-11, or ≥12years of age, respectively.

In general, one of ordinary skill in the art can select an appropriatemeans of determining asthma severity level and/or degree of control, andany classification scheme considered reasonable by those of ordinaryskill in the art can be used.

In some embodiments of the invention, a subject suffering frompersistent asthma is treated with a complement inhibitor using aninventive dosing regimen. In some embodiments, the subject suffers frommild or moderate asthma. In some embodiments, the subject suffers fromsevere asthma. In some embodiments, a subject has asthma that is notwell controlled using conventional therapy. In some embodiments, asubject has asthma that, when treated using conventional therapy,requires use of ICS in order to be well controlled. In some embodiments,a subject has asthma that fails to be well controlled despite use ofICS. In some embodiments, a subject has asthma that, if treated usingconventional therapy, would require use of OCS in order to be wellcontrolled. In some embodiments, a subject has asthma that fails to bewell controlled despite use of high intensity conventional therapy thatincludes OCS. In some embodiments of the invention a long-actingcomplement inhibitor is administered as a controller medication or allowthe subject to avoid using or reduce their dose of a conventionalcontroller medication.

In some embodiments, the subject suffers from allergic asthma, which isthe case for most asthmatic individuals. In some embodiments, anasthmatic subject is considered to have allergic asthma if anon-allergic trigger for the asthma (e.g., cold, exercise) is not knownand/or is not identified in a standard diagnostic evaluation. In someembodiments, an asthmatic subject is considered to have allergic asthmaif the subject (i) reproducibly develops asthma symptoms (or worseningof asthma symptoms) following exposure to an allergen or allergen(s) towhich the subject is sensitive; (ii) exhibits IgE specific for anallergen or allergen(s) to which the subject is sensitive; (iii)exhibits a positive skin-prick test to an allergen or allergen(s) towhich the subject is sensitive; and/or (iv) exhibits other symptom(s) ofcharacteristic(s) consistent with atopy such as allergic rhinitis,eczema, or elevated total serum IgE. It will be appreciated that aspecific allergic trigger may not be identified but may be suspected orinferred if the subject experiences worsening symptoms in particularenvironments, for example.

Allergen challenge by inhalation is a technique that is widely used inevaluating allergic airway disease. Inhalation of allergen leads tocross-linking of allergen-specific IgE bound to IgE receptors on, e.g.,mast cells and basophils. Activation of secretory pathways ensues,resulting in release of mediators of bronchoconstriction and vascularpermeability. Individuals with allergic asthma may develop variousmanifestations following allergen challenge, e.g., early asthmaticresponse (EAR), late asthmatic response (LAR), airway hyperreactivity(AHR), and airway eosinophilia, each of which can be detected andquantified as known in the art. For example, airway eosiphophilia may bedetected as an increase in eosinophils in sputum and/or BAL fluid. TheEAR, sometimes referred to as the immediate asthmatic response (IAR), isa response to allergen challenge by inhalation that becomes detectableshortly after the inhalation, typically within 10 minutes (min) of theinhalation, e.g., as a decrease in FEV₁. The EAR typically reaches amaximum within 30 min and resolves within 2-3 hours (h) post-challenge.For example, a subject may be considered to exhibit a “positive” EAR ifhis/her FEV₁ decreases by at least 15%, e.g., at least 20%, within thistime window relative to baseline FEV₁ (where “baseline” in this contextrefers to conditions before the challenge, e.g., conditions equivalentto the subject's usual condition when not experiencing an asthmaexacerbation and not exposed to allergic stimuli to which the subject issensitive). The late asthmatic response (LAR) typically starts between 3h and 8 h post-challenge and is characterized by cellular inflammationof the airway, increased bronchiovascular permeability, and mucussecretion. It is typically detected as a decrease in FEV₁, which may begreater in magnitude than that associated with the EAR and potentiallymore clinically important. For example, a subject may be considered toexhibit a “positive” LAR if his/her FEV₁ decreases by at least 15%,e.g., at least 20%, relative to baseline FEV₁ within the relevant timeperiod as compared with baseline FEV₁. A delayed airway response (DAR)may occur beginning between about 26 and 32 h, reaching a maximumbetween about 32 and 48 h and resolving within about 56 h after thechallenge (Pelikan, Z. Ann Allergy Asthma Immunol. 2010,104(5):394-404).

In some embodiments, the chronic respiratory disorder is chronicobstructive pulmonary disease (COPD). COPD encompasses a spectrum ofconditions characterized by airflow limitation that is not fullyreversible even with therapy and is usually progressive. Symptoms ofCOPD include dyspnea (breathlessness), decreased exercise tolerance,cough, sputum production, wheezing, and chest tightness. Persons withCOPD can experience episodes of acute (e.g., developing over course ofless than a week and often over the course of 24 hours or less)worsening of symptoms (termed COPD exacerbations) that can vary infrequency and duration and are associated with significant morbidity.They may be triggered by events such as respiratory infection, exposureto noxious particles, or may have an unknown etiology. Smoking is themost commonly encountered risk factor for COPD, and other inhalationalexposures can also contribute to development and progression of thedisease. The role of genetic factors in COPD is an area of activeresearch. A small percentage of COPD patients have a hereditarydeficiency of alpha-1 antitrypsin, a major circulating inhibitor ofserine proteases, and this deficiency can lead to a rapidly progressiveform of the disease.

Characteristic pathophysiologic features of COPD include narrowing ofand structural changes in the small airways and destruction of lungparenchyma (in particular around alveoli), most commonly due to chronicinflammation. The chronic airflow limitation observed in COPD typicallyinvolves a mixture of these factors, and their relative importance incontributing to airflow limitation and symptoms varies from person toperson. The term “emphysema” refers to enlargement of the air spaces(alveoli) distal to the terminal bronchioles, with destruction of theirwalls. It should be noted that the term “emphysema” is often usedclinically to refer to the medical condition associated with suchpathological changes. Some individuals with COPD have chronicbronchitis, which is defined in clinical terms as a cough with sputumproduction on most days for 3 months of a year, for 2 consecutive years.Further information regarding risk factors, epidemiology, pathogenesis,diagnosis, and current management of COPD may be found, e.g., in “GlobalStrategy for the Diagnosis, Management, and Prevention of ChronicObstructive Pulmonary Disease” (updated 2009) available on the GlobalInitiative on Chronic Obstructive Pulmonary Disease, Inc. (GOLD) website(www.goldcopd.org), also referred to herein as the “GOLD Report”, theAmerican Thoracic Society/European Respiratory Society Guidelines (2004)available on the ATS website atwww.thoracic.org/clinical/copd-guidelines/resources/copddoc.pdf,referred to herein as “ATC/ERS COPD Guidelines” and standard textbooksof internal medicine such as Cecil Textbook of Medicine (20^(th)edition), Harrison's Principles of Internal Medicine (17^(th) edition),and/or standard textbooks focusing on pulmonary medicine.

In some embodiments methods disclosed herein inhibit (interfere with,disrupt) the DC-Th17-B-Ab-C-DC cycle discussed above. For example,administration of a complement inhibitor may break the cycle by whichcomplement stimulates DC cells to promote the Th17 phenotype. As aresult, the number and/or activity of Th17 cells diminishes, which inturn reduces the amount of Th17-mediated stimulation of B cells andpolyclonal antibody production. In some embodiments, these effectsresult in “resetting” the immunological microenvironment to a morenormal, less pathological state. As described in Example 1, evidencesupporting the capacity of complement inhibition to have a prolongedinhibitory effect on Th17-associated cytokine production was obtained inan animal model of asthma.

In some embodiments, inhibiting the DC-Th17-B-Ab-C-DC cycle has adisease-modifying effect. Without wishing to be bound by any theory,rather than merely treating symptoms of a disorder, inhibiting theDC-Th17-B-Ab-C-DC cycle may interfere with fundamental pathologicmechanisms that may contribute to ongoing tissue damage even whensymptoms are well controlled and/or that may contribute to exacerbationsof the disease. In some embodiments, inhibiting the DC-Th17-B-Ab-C-DCcycle causes a chronic disorder to go into remission. In someembodiments, remission refers to a state of absence or substantialabsence of disease activity in a subject with a chronic disorder, withthe possibility of return of disease. In some embodiments remission maybe sustained for a prolonged period of time (e.g., at least 6 months,e.g., 6-12 months, 12-24 months, or more) in the absence of continuedtherapy or with a reduced dose or increased dosing interval. In someaspects, inhibition of complement may change the immunologicalmicro-environment of a tissue that is rich in Th17 cells and modify itinto a micro-environment that is rich in regulatory T cells (Tregs).Doing so could allow the immune system to “reset” itself and go into astate of remission. In some embodiments, for example, remission may besustained until occurrence of a triggering event. A triggering event maybe, for example, an infection (which may result in production ofpolyclonal antibodies that react both with an infectious agent and aself protein), exposure to particular environmental conditions (e.g.,high levels of air pollutants such as ozone or particulate matter orcomponents of smoke such as cigarette smoke, allergens), etc. Geneticfactors may play a role. For example, individuals having particularalleles of genes encoding complement components may have a higherbaseline level of complement activity, a more reactive complement systemand/or a lower baseline level of endogenous complement regulatoryprotein activity. In some embodiments an individual has a genotypeassociated with increased risk of AMD. For example, the subject may havea polymorphism in a gene encoding a complement protein or complementregulatory protein, e.g., CFH, C3, factor B, wherein the polymorphism isassociated with an increased risk of AMD.

In some embodiments an immunologic microenvironment may becomeprogressively more polarized towards a pathological state over time,e.g., in a subject who has not yet developed symptoms of a chronicdisorder or in a subject who has developed the disorder and has beentreated as described herein. Such a transition may occur stochastically(e.g., due at least in part to apparently random fluctuations inantibody levels and/or affinity) and/or as a result of accumulated“sub-threshold” trigger events that are not of sufficient intensity totrigger a symptomatic outbreak of a disorder.

In some embodiments it is contemplated that a relatively short course ofa long-acting compstatin analog, e.g., between 1 week and 6 weeks, e.g.,about 2-4 week, may provide a long-lasting benefit. In some embodimentsa remission is achieved for a prolonged period of time, e.g., 1-3months, 3-6 months, 6-12 months, 12-24 months, or more. In someembodiments a subject may be monitored and/or treated prophylacticallybefore recurrence of symptoms. For example, a subject may be treatedprior to or upon exposure to a triggering event. In some embodiments asubject may be monitored, e.g., for an increase in a biomarker, e.g., abiomarker comprising an indicator of Th17 cells or Th17 cell activity,or complement activation, and may be treated upon increase in the levelof such biomarker. See, e.g., PCT/US2012/043845 for further discussion.

IX. Compositions and Administration

The invention provides a variety of compositions comprising acell-reactive, long-acting, or targeted compstatin analog. In variousembodiments, a composition can have any feature or combination offeatures discussed herein so long as they are not mutually exclusive.The invention provides embodiments of such compositions, and methods ofuse thereof, in which the compstatin analog is any compstatin analog.

In some embodiments, a composition comprises a purified cell-reactive,long-acting, or targeted compstatin analog. Purification can be achievedusing a variety of approaches that can be selected by one of ordinaryskill in the art based to achieve a desired degree of purity withrespect to various components present in the composition prior topurification. For example, filtration, high performance liquidchromatography, affinity chromatography, and/or other approaches andcombinations thereof can be used. In some embodiments, the compositioncomprises at least 80%, 85%, 90%, 95%, 98%, 99%, or more cell-reactive,long-acting, or targeted compstatin analog as a percentage of the totalcompstatin analog by weight. In some embodiments, the compositioncomprises at least 80%, 85%, 90%, 95%, 98%, 99%, or more cell-reactive,long-acting, or targeted compstatin analog as a percentage of the totalcompstatin analog on a molar basis. In some embodiments, a compositionconsists or consists essentially of a cell-reactive, long-acting, ortargeted compstatin analog.

In some embodiments, a composition comprising a cell-reactive compstatinanalog and a compound comprising a cell-reactive functional group ischaracterized in that the ratio of the cell-reactive compstatin analogto the compound comprising the cell-reactive functional group on a molarbasis is at least 10:1, 20:1, 50:1, 100:1, 500:1, 1,000:1, or more. Insome embodiments the composition comprises at least 80%, 85%, 90%, 95%,98%, 99%, or more cell-reactive compstatin analog as a percentage of thetotal compstatin analog by weight. In some embodiments the compositioncomprises at least 80%, 85%, 90%, 95%, 98%, 99%, or more cell-reactivecompstatin analog as a percentage of the total compstatin analog on amolar basis. In some embodiments a composition comprises at least 80%,85%, 90%, 95%, 98%, 99%, or more cell-reactive compstatin analog byweight. In some embodiments a composition comprises at least 80%, 85%,90%, 95%, 98%, 99%, or more long-acting compstatin analog by weight. Insome embodiments a composition comprises at least 80%, 85%, 90%, 95%,98%, 99%, or more targeted compstatin analog by weight. In someembodiments a composition comprises at least 80%, 85%, 90%, 95%, 98%,99%, or more targeted compstatin analog by weight. In some embodimentsweight is dry weight.

In some aspects, the invention provides a pharmaceutical gradecomposition comprising a cell-reactive, long-acting, or targetedcompstatin analog. The pharmaceutical grade composition can have any ofthe above-mentioned characteristics in terms of purity in variousembodiments. The pharmaceutical grade composition is sufficiently freeof endotoxin, heavy metals, and unidentified and/or uncharacterizedsubstances so as to be acceptable, without further purification, as apharmaceutical composition suitable for administration to a humansubject or for the manufacture of a pharmaceutical composition to beadministered to a human subject. In some embodiments, the pharmaceuticalgrade composition is sterile.

Suitable preparations, e.g., substantially pure preparations of acell-reactive, long-acting, or targeted compstatin analog or otheractive agent, may be combined with pharmaceutically acceptable carriersor vehicles, etc., to produce an appropriate pharmaceutical composition.The term “pharmaceutically acceptable carrier or vehicle” refers to anon-toxic carrier or vehicle that does not destroy the pharmacologicalactivity of the compound with which it is formulated. One of skill inthe art will understand that a carrier or vehicle is “non-toxic” if itis compatible with administration to a subject in an amount appropriateto deliver the compound without causing undue toxicity. Pharmaceuticallyacceptable carriers or vehicles that may be used in the compositions ofthis invention include, but are not limited to, water, physiologicalsaline, Ringer's solution, sodium acetate or potassium acetate solution,5% dextrose, and the like. The composition may include other componentsas appropriate for the formulation desired, e.g., as discussed herein.Supplementary active compounds, e.g., compounds independently useful fortreating a subject suffering from a complement-mediated disorder, canalso be incorporated into the compositions. The invention provides suchpharmaceutical compositions comprising a cell-reactive, long-acting, ortargeted compstatin analog and, optionally, a second active agent usefulfor treating a subject suffering from a complement-mediated disorder.

In some embodiments, the invention provides a pharmaceuticallyacceptable composition suitable for administration to humans, packagedtogether with a label approved by a government agency responsible forregulating pharmaceutical agents, e.g., the U.S. Food & DrugAdministration. In some embodiments, the invention provides apharmaceutical kit or pack comprising: (a) a pharmaceutically acceptablecell-reactive, long-acting, or targeted compstatin analog in solid form;(b) a pharmaceutically acceptable carrier or vehicle. Optionally the kitor pack contains instructions for dissolving the cell-reactive,long-acting, or targeted compstatin analog in the carrier. In someembodiments a pharmaceutical kit or pack is provided. The pack or kitcomprises sufficient amount of pharmaceutical composition for at least 1dose, e.g., between 1 and 200 doses or any intervening number orsubrange. In some embodiments a pharmaceutical pack or kit comprises oneor more needles and, optionally, one or more syringes. In someembodiments at least one prefilled syringe is provided. In someembodiments one or more unit dosage forms or premeasured aliquots areprovided. In some embodiments instructions for administration, which insome embodiments comprise instructions for self-administration, e.g.,via subcutaneous injection, are provided.

A pharmaceutical composition can be administered to a subject by anysuitable route of administration including, but not limited to,intravenous, intramuscular, subcutaneously, by inhalation, by nasaldelivery, intrathecally, intracranially, intraarterially, orally,rectally, transdermally, intradermally, subdermally, etc. In someembodiments, a composition comprising a cell-reactive, long-acting, ortargeted compstatin analog is administered intravenously. In someembodiments, a composition comprising a cell-reactive, long-acting, ortargeted compstatin analog is administered intra-arterially. Thecomposition can be administered locally, either into the vascular systemsupplying an organ or tissue, or extra-vascularly in the vicinity of anorgan or tissue. It will be understood that “administration” encompassesdirectly administering a compound or composition to a subject,instructing a third party to administer a compound or composition to asubject, prescribing or suggesting a compound or composition to asubject (e.g., for self-administration), self-administration, and, asappropriate, other means of making a compound or composition availableto a subject.

Pharmaceutical compositions suitable for injectable use (e.g.,intravenous administration) or by pump or catheter typically includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. Sterile solutions can be prepared byincorporating the compound in the required amount in an appropriatesolvent, optionally with one or a combination of ingredients such asbuffers such as acetates, citrates, lactates or phosphates; agents forthe adjustment of tonicity such as sodium chloride or dextrose;antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid, glutathione, or sodium bisulfate;chelating agents such as ethylenediaminetetraacetic acid; and othersuitable ingredients etc., as desired, followed by filter-basedsterilization. One of skill in the art will be aware of numerousphysiologically acceptable compounds that may be included in apharmaceutical composition. Other useful compounds include, for example,carbohydrates, such as glucose, sucrose, lactose; dextrans; amino acidssuch as glycine; polyols such as mannitol. These compounds may, forexample, serve as bulking agents and/or stabilizers, e.g., in a powderand/or when part of the manufacture or storage process involveslyophilization. Surfactant(s) such as Tween-80, Pluronic-F108/F68,deoxycholic acid, phosphatidylcholine, etc., may be included in acomposition, e.g., to increase solubility or to provide microemulsion todeliver hydrophobic drugs. pH can be adjusted with acids or bases, suchas hydrochloric acid or sodium hydroxide, if desired. The parenteralpreparation can be enclosed in ampoules, disposable syringes or infusionbags or multiple dose vials made of glass or plastic. Preferablysolutions for injection are sterile and acceptably free of endotoxin.

Generally, dispersions are prepared by incorporating the active compoundinto a sterile vehicle which contains a basic dispersion medium andappropriate other ingredients from those enumerated above. In the caseof sterile powders for the preparation of sterile injectable solutions,methods of preparation can include vacuum drying and freeze-drying whichyields a powder of the active ingredient plus any additional desiredingredient, e.g., from a previously sterile-filtered solution thereof.

Oral administration may be used in certain embodiments. Oralcompositions generally include an inert diluent or an edible carrier.For the purpose of oral therapeutic administration, the active compoundcan be incorporated with excipients and used in the form of tablets,troches, or capsules, e.g., gelatin capsules. Pharmaceuticallycompatible binding agents; and/or adjuvant materials can be included aspart of the composition. The tablets, pills, capsules, troches and thelike can contain any of the following ingredients, or compounds of asimilar nature: a binder such as microcrystalline cellulose, gumtragacanth or gelatin; an excipient such as starch or lactose, adisintegrating agent such as alginic acid, Primogel, or corn starch; alubricant such as magnesium stearate or Sterotes; a glidant such ascolloidal silicon dioxide; a sweetening agent such as sucrose orsaccharin; or a flavoring agent such as peppermint, methyl salicylate,or orange flavoring. A liquid composition can also be administeredorally. Formulations for oral delivery may incorporate agents to improvestability within the gastrointestinal tract and/or to enhanceabsorption.

For administration by inhalation, a compstatin analog may be deliveredin the form of an aerosol spray from a pressured container or dispenserwhich contains a suitable propellant, e.g., a gas such as carbondioxide. A metered dose inhaler or nebulizer may be used. The aerosolmay comprise liquid particles or dry aerosol (e.g., dry powders, largeporous particles, etc.).

For topical application, a compstatin analog may be formulated in asuitable ointment containing the active component suspended or dissolvedin one or more carriers. Carriers for topical administration include,but are not limited to, mineral oil, liquid petrolatum, whitepetrolatum, propylene glycol, polyoxyethylene, poly oxypropylenecompound, emulsifying wax and water. Alternatively, the pharmaceuticallyacceptable compositions can be formulated as a suitable lotion or creamcontaining a compstatin analog suspended or dissolved in one or morepharmaceutically acceptable carriers. Suitable carriers include, but arenot limited to, mineral oil, sorbitan monostearate, polysorbate 60,cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol,and water.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated may be used in theformulation. Such penetrants are generally known in the art, andinclude, for example, for transmucosal administration, detergents, bilesalts, and fusidic acid derivatives. Transmucosal administration can beaccomplished, e.g., through the use of nasal sprays or suppositories.For transdermal administration, the active compounds are typicallyformulated into ointments, salves, gels, or creams as generally known inthe art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In certain embodiments of the invention, a compstatin analog or otheractive compound is prepared with carriers that will protect the compoundagainst rapid elimination from the body, such as a controlled. releaseformulation, including; implants and microencapsulated delivery systems.For example, a compstatin analog may be incorporated into orencapsulated in a microparticle or nanoparticle formulation.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, polyethers, polylactic acid, PLGA, etc. Liposomes orother lipid-based particles can be used as pharmaceutically acceptablecarriers. These can be prepared according to methods known to thoseskilled in the art, for example, as described in U.S. Pat. No. 4,522,811and/or other references listed herein. Depot formulations containing acompstatin analog may be used. The compstatin analog is released fromthe depot over time, e.g., so as to provide a therapeutic concentrationfor longer than if the compound was administered intravenously. In someaspects, a CRM confers depot properties on a compstatin analog. One ofordinary skill in the art will appreciate that the materials and methodsselected for preparation of a controlled release formulation, implant,etc., should be such as to retain activity of the compound.

It will be appreciated that the compstatin analog and/or additionalactive agent(s) can be provided as a pharmaceutically acceptable salt.Pharmaceutically acceptable salts include those derived frompharmaceutically acceptable inorganic and organic acids and bases.Examples of suitable acid salts include acetate, adipate, alginate,aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate,camphorate, camphorsulfonate, cyclopentanepropionate, digluconate,dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate,glycerophosphate, glycolate, hemisulfate, heptanoate, hexanoate,hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate,lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate,nicotinate, nitrate, oxalate, palmoate, pectinate, persulfate,3-phenylpropionate, phosphate, picrate, pivalate, propionate,salicylate, succinate, sulfate, tartrate, thiocyanate, tosylate andundecanoate. Also, pharmaceutically-acceptable salts can be prepared asalkaline metal or alkaline earth salts, such as sodium, potassium orcalcium salts, if appropriate depending on the identity of the activeagent.

It will be understood that the pharmaceutically acceptable carriers,compounds, and preparation methods mentioned herein are exemplary andnon-limiting. See, e.g., Remington: The Science and Practice ofPharmacy. 21st Edition. Philadelphia, Pa. Lippincott Williams & Wilkins,2005, for additional discussion of pharmaceutically acceptable compoundsand methods of preparing pharmaceutical compositions of various types.

A pharmaceutical composition can be administered in an amount effectiveto achieve a desired beneficial effect. In some embodiments, aneffective amount is sufficient to provide one or more of the followingbenefits: (i) reduction in at least one symptom or sign of acomplement-mediated disorder; (ii) increased quality of life; (iii)reduced hospitalization; (iv) reduced mortality. One of ordinary skillin the art will appreciate that the particular beneficial effect willdepend at least in part on various factors, such as the particulardisorder being treated. One of ordinary skill in the art will be awareof the symptoms and signs that may occur in subjects withcomplement-mediated disorders. Examples of symptoms and signs of variouscomplement disorders are provided herein. For example, in someembodiments, e.g., wherein a subject suffers from PNH or aHUS, abeneficial effect is a reduction in complement-mediated red blood celllysis. In some aspects, a beneficial effect is statistically significantand/or therapeutically meaningful within the judgement of one orordinary skill in the art.

In certain embodiments of the invention a pharmaceutical compositioncomprising a cell-reactive, long-acting, or targeted compstatin analogis administered parenterally. In some embodiments, the composition isadministered intravenously. In some embodiments, the composition isadministered by intravenous injection. In some embodiments thecomposition is administered as an IV bolus or an IV infusion. In someembodiments the composition is administered as an IV drip. In someembodiments the composition is administered as an IV bolus followed byan IV infusion or IV drip. In some embodiments an IV infusion isadministered over about 1, 2, 3, 4, 5, 15, 20, 30, 60, or 120 minutes.In some embodiments an IV drip is administered over more than about 60minutes, e.g., over about 1, 2, 3, or more hours. In some embodiments, atotal amount of between about 0.1 mg/kg/day and about 2,000 mg/kg/day ofcompstatin analog is administered, e.g., between about 1 mg/kg/day andabout 1,000 mg/kg/day, e.g., between about 5 mg/kg/day and about 500mg/kg/day. In some embodiments, a total amount of between about 10mg/kg/day and about 100 mg/kg/day of compstatin analog is administered,e.g., between about 10 mg/kg/day and about 50 mg/kg/day e.g., betweenabout 10 mg/kg/day and about 20 mg/kg/day. In some embodiments, betweenabout 0.5 mg/kg/day to about 10 mg/kg/day of compstatin analog isadministered. In some embodiments, between about 1 mg/kg/day to about 5mg/kg/day of compstatin analog is administered. In some embodiments,between about 1 mg/kg/day to about 3 mg/kg/day of compstatin analog isadministered. In some embodiments, between about 3 mg/kg/day to about 5mg/kg/day of compstatin analog is administered. In some embodiments,between about 5 mg/kg/day to about 7.5 mg/kg/day of compstatin analog isadministered. In some embodiments, between about 7.5 mg/kg/day to about10 mg/kg/day of compstatin analog is administered. It will beappreciated that a variety of different dosing regimens could be used toadminister a desired total daily amount. For example, a desired amountof compstatin analog could be administered in a single administration orin multiple administrations, e.g., during a 24 hour period. For example,a subject could receive two or more doses within a 24 hour period, whichdoses could be administered over the same length of time or overdifferent lengths of time. In some embodiments, a cell-reactive,long-acting, or targeted compstatin analog is administered at timeintervals greater than 24 hours. For example, doses could beadministered on average every other day, every 3-4 days, weekly, everyother week, etc., in various embodiments. In some embodiments,covalently attached, long-acting, or targeted compstatin analogs protectcells, tissues, organs, for a period of weeks or months without need forretreatment. For example, subjects may be maintained with retreatment atintervals of between 1-2 weeks, 2-4 weeks, 4-6 weeks, 6-8 weeks, or evenlonger. In some embodiments subcutaneous administration is used toadminister at least some doses. For example, administration ofapproximately 0.1-5 mg/kg/day, e.g., about 0.5-2 mg/kg/day iscontemplated in some embodiments, e.g., in a volume of about 0.25 ml-2mL, e.g., a volume of about 1 ml. In some embodiments the concentrationis about 50 mg/ml to about 300 mg/ml, e.g., about 50 mg/ml-about 100mg/ml or about 100 mg/ml-about 200 mg/ml. In some embodimentsadministration is daily. In some embodiments administration is 1 or 2times per day. As described further in the Examples, daily subcutaneousadministration of an exemplary long-acting compstatin analog readilyachieved blood levels well above 5 micromolar. In some embodiments,intramuscular administration is used to deliver similar amounts ofcompound. In some embodiments a long-acting compstatin analog isadministered using a therapeutically effective amount to a subject,wherein such administration results in blood concentrations of thecompound that achieve a level above at least 1 μM, at least 2 μM, atleast 2.5 μM, at least 3 μM, at least 4 μM, at least 5 μM, at least 6μM, at least 7 μM, at least 8 μM, at least 9 μM, at least 10 μM, atleast 11 μM, at least 12 μM, or at least 13 μM, at least 14 μM, at least15 μM, at least 16 μM, at least 18 μM, or at least about 20 μM, or atleast about 25 μM or within any range between 4 μM and about 15 μM orabout 20 μM or about 25 μM. In some embodiments such level is maintainedfor at least about 24 hours, or at least about 48 hours, or at leastabout 72 hours, or at least about 96 hours, or at least about 120 hours,or at least about 144 hours following a single IV injection or followingdaily subcutaneous injections for about 5-7 days. Sustained levels maybe achieved for longer, e.g., up to about 10 days, 12 days, 14 days, ormore. In some embodiments a subject is treated so as to maintain asteady state level of about 1.0 μM, about 2.0 μM, about 2.5 μM, about3.0 μM, about 3.5 μM, about 4.0 μM, about 4.5 μM, about 5.0 μM, about5.5 μM, about 6.0 μM, about 6.5 μM, about 7.0 μM, about 7.5 μM, about8.0 μM, about 8.5 μM, about 9.0 μM, about 9.5 μM, or about 10 μM. Insome embodiments a steady state level has a value between about 1.0 μMand about 10.0 μM, e.g., between about 2.0 μM and about 5.0 μM, betweenabout 2.5 μM and about 5.0 μM, between about 5.0 μM and about 7.5 μM, orbetween about 7.5 μM, and about 10 μM, or any intervening value withinany of the afore-mentioned ranges. In some embodiments a concentrationis sufficient to substantially inhibit lysis of red blood cells of PNHpatients exposed to human serum in vitro, e.g., using a modified Ham'sassay using human serum (see, e.g., Example 8). In some embodiments aconcentration is sufficient to reduce by at least 50%, 60%, 70%, 80%,90%, or more, lysis of red blood cells of PNH patients exposed to humanserum in vitro, e.g., using a modified Ham's assay using human serum(see, e.g., Example 8). In some embodiments a Ham's assay may beperformed using human serum adjusted to a magnesium level at about 0.005mol/L and a pH lowered to about 6.2 to activate complement. Examples 18and 19 present data confirming the ability of compstatin analogsdescribed herein to inhibit lysis of RBCs from PNH patients.

In some aspects compstatin analogs, e.g., long-acting compstatinanalogs, may protect red blood cells of PNH patients from accumulatingsignificant amounts of C3 and/or products of C3 activation on theirsurface. For example, PNH red blood cells that are protected fromcomplement-mediated lysis by compstatin analogs, e.g., long-actingcompstatin analogs, may also be protected from accumulating significantamounts of C3 and/or products of C3 activation on their surface. Asknown in the art, eculizumab (Soliris®, Alexion Pharmaceuticals. Inc.),is a humanized anti-C5 monoclonal antibody that is approved fortreatment of PNH and aHUS in a number of countries (see, e.g., DmytrijukA, FDA report: eculizumab (Soliris) for the treatment of patients withparoxysmal nocturnal hemoglobinuria. Oncologist. 2008 September;13(9):993-1000. doi: 10.1634/theoncologist.2008-0086. Epub 2008 Sep. 10;Westra D., A new era in the diagnosis and treatment of atypicalhaemolytic uraemic syndrome. Neth J Med. 2012 April; 70(3):121-9). Ithas been reported that when PNH RBCs are exposed to eculizumab they mayexhibit accumulation of significant amounts of C3 and/or products of C3activation on their surface, which may contribute to the clearanceand/or extravascular hemolysis of these cells (e.g., in the spleen) andmay thus at least in part account for persistent hematologicabnormalities, e.g., persistent anemia, observed in some PNH patientsdespite treatment with eculizumab. Without wishing to be bound by anytheory, this may occur due to inhibition of formation of the MAC byeculizumab, which protects cells from MAC-mediated lysis but does notinhibit C3 activation or deposition of C3 and/or products of C3activation and leaves PNH cells vulnerable to surface C3 activation anddeposition of C3 and/or products of C3 activation due to their lack ofGPI-anchored complement inhibiting proteins. Without wishing to be boundby any theory, the ability of compstatin analogs described herein toinhibit C3 activation and thereby inhibit the production of C3activation products may afford a significant advantage. In someembodiments a subject who has been or is being treated with eculizumaband continues to exhibit evidence of hemolysis, e.g., clinicallysignificant hemolysis, such as causing anemia and/or requiringtransfusion is treated with a compstatin analog described herein. Insome embodiments a compstatin analog is used at a concentrationsufficient such that the level of C3 and/or C3 activation products onPNH RBCs exposed to the compstatin analog (in vitro (e.g., in a Ham'sassay) or in vivo) is within the range exhibited by normal RBCs fromhealthy subjects. In some embodiments the level of C3 and/or C3activation products on PNH RBCs exposed to the compstatin analog (invitro (e.g., in a Ham's assay) or in vivo) is within about 1.2, 1.5,2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 of the average level or upper limitof normal. In some embodiments the level of C3 and/or C3 activationproducts on PNH RBCs exposed to a compstatin analog (in vitro (e.g., ina Ham's assay) or in vivo) is less than the level of C3 and/or C3activation products on PNH RBCs exposed to Soliris at a concentrationthat provides equivalent protection against complement-mediated lysis.In some embodiments the level of C3 and/or C3 activation products on PNHRBCs exposed to a compstatin analog (in vitro or in vivo) is no morethan about 5%, 10%, 15%, 20%, 25%, 30%, 40%, or 50% of the level of C3and/or C3 activation products on PNH RBCs exposed to eculizumab at aconcentration that provides equivalent protection againstcomplement-mediated lysis. In some embodiments the level of C3 and/or C3activation products on PNH RBCs exposed to the compstatin analog (invitro or in vivo) is within about 1.2, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0,4.5, or 5.0 of the average level or upper limit of a normal range. Insome embodiments the PNH cells comprise or consist of Type II PNH cells,Type III PNH cells, or a mixture thereof. In some embodiments the RBCsare at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more Type IIIand/or Type II RBCs. In some embodiments the cells may comprise someType I cells. In some embodiments RBCs may be classified as Type I, II,or III based on the level of a GPI-anchored protein such as CD59 ontheir surface, which may be measured using flow cytometry,immunofluorescence, or ELISA, e.g., using an antibody (e.g., amonoclonal antibody) or other binding agent that binds to saidGPI-anchored protein. In some aspects inhibition of deposition of C3and/or products of C3 activation on cells or surfaces may be used as anindicator of efficacy of a compstatin analog in othercomplement-mediated diseases such as aHUS, other complement-mediatedhemolytic diseases, or other complement-mediated diseases. For example,in some embodiments a compstatin analog inhibits such deposition onendothelial cells in a subject with aHUS. In some embodiments the levelof C3 and/or C3 activation product(s) may be measured using flowcytometry, immunofluorescence, or ELISA, e.g., using an antibody (e.g.,a monoclonal antibody) or other binding agent that binds to C3 and/or toone or more C3 activation product(s). In some embodiments a C3activation product is C3b, C3c, or C3d. In some embodiments a bindingagent binds to C3d. In some embodiments a binding agent binds to C3d andat least one other C3 activation product. In some embodiments PNHpatient RBCs contacted with a compstatin analog in vitro (e.g., in aHam's assay) are protected from activated complement such that therelative proportion (percentages) of Type I, Type II, and Type III cellsor the relative proportion or percentages of Type III and Type I, TypeII and Type I, or Type III and Type II, are approximately the same as ina control assay in which inactivated complement (e.g., heat inactivatedcomplement) is used. In some embodiments PNH patient RBCs contacted witha compstatin analog in vitro (e.g., in a Ham's assay) are protected fromactivated complement such that the relative proportion or percentages ofType I, Type II, and Type III cells or the relative proportion orpercentages of Type III and Type I, Type II and Type I, or Type III andType II, are within 5% of the proportions or percentages obtained in acontrol assay in which inactivated complement (e.g., heat inactivatedcomplement) is used. In some embodiments complement may be inactivatedby heat inactivation, which may be performed by heating complementcomponents or serum or plasma containing complement components to 56degrees C. or higher.

In some embodiments measurements of LDH (an enzyme that is abundant inred blood cells and can function as a marker for hemolysis), one or morehematologic parameters such as hematocrit, hemoglobin, and/orreticulocyte measurements may additionally or alternately be used indetermining the amount of lysis. In some embodiments one or more suchmethods may be used to determine the amount of lysis of RBCs, e.g., RBCsthat are susceptible to complement-mediated lysis, e.g., PNH patientcells, aHUS patient cells, cells from subjects with othercomplement-mediated hematologic disorders, cells exposed to abnormallyhigh levels of complement activation. In some aspects, the disclosureprovides a method comprising contacting one or more cells in vitro or invivo with a compstatin analog described herein and measuring the effectof the compstatin analog on one or more indicators ofcomplement-mediated cell damage and/or cell surface complementactivation or deposition. In some embodiments contacting the one or morecells for a sufficient time at a sufficient concentration results in areduction of an abnormally high value or an increase in an abnormallylow value to within a normal range or to within 5%, 10%, 15%, 20%, or25% of the lower or upper limit of a normal range.

In some aspects, the disclosure provides a method of selecting ormodifying a dosing regimen or one or more components of a dosing regimenfor a patient with a complement-mediated hemolytic disease such as PNH.The one or more components of a dosing regimen may comprise a dose,dosing interval, route of administration (e.g., IV or subcutaneous), orcombination thereof. A dose may be a loading dose, maintenance dose, orboth. In some embodiments, one or more blood samples may be obtainedfrom a patient and a dosing regimen or component thereof for acompstatin analog, e.g., a long-acting compstatin analog, may beselected or modified to achieve a desired level of protection of thepatient's RBCs from lysis and/or from accumulation of C3 and/or C3activation product(s) in vitro. In some embodiments one or more doses ofa compstatin analog, e.g., a long-acting compstatin analog, may beadministered to a patient, and one or more blood samples may besubsequently obtained from a patient and assessed for level of C3 and/orC3 activation product(s) on their surface. In some embodiments a dosingregimen or component thereof, e.g., a dose, dosing interval, or route ofadministration, may be selected or modified to achieve a desired levelof protection of the patient's RBCs from lysis and/or from accumulationof C3 and/or C3 activation products in vitro or in vivo. In someembodiments a dosing regimen or component thereof, e.g., dose, dosinginterval, or route of administration, may be selected or modified toachieve a desired level of protection of the patient's RBCs fromextravascular clearance and/or extravascular lysis in vivo. A desiredlevel may be, e.g., a level that is accepted in the art as providing aclinically meaningful benefit, a level that provides a clinicallymeaningful benefit to a particular patient, a level that is within thenormal range, a level selected by a medical practitioner, or any otherselected level. A normal range for a parameter may be known in the artand/or may be a reference range established by a laboratory, e.g., aclinical laboratory, wherein the value of the relevant parameter asmeasured in at least 95%, 96%, 97%, 98%, or 99% of the generalpopulation or at least 95%, 96%, 97%, 98%, or 99% of healthy individuals(which may optionally be matched for one or more demographic variablessuch as gender, age, etc.) or biological specimens obtained therefrom(such as blood samples) would fall within the reference range. Areference range may be established using a sample populationrepresentative of the general population or representative of healthyindividuals.

In some embodiments a long-acting compstatin analog comprising a CRM isdesigned to confer a slower rate of systemic absorption aftersubcutaneous or intramuscular administration to a subject as comparedwith a compstatin analog not comprising a CRM. In some embodimentsparticular CRM properties, e.g., length, are selected to confer adesired rate of systemic absorption after subcutaneous or intramuscularadministration as compared with at least some other CRMs. In someembodiments, the Cmax is reduced in comparison to a comparable dose of acompstatin analog not linked to a CRM, which may thereby contribute tokeeping the plasma concentration within a desired window, e.g., thetherapeutic window, for the compound. In some embodiments a long-actingcompstatin analog composition is characterized in that a dose, whenadministered subcutaneously, appears fully absorbed within about 1, 2,3, 4, 6, 8, 12, 15, 30, 45, 60, 90, or 120 hours followingadministration based on visual observation at the injection site.

It will be understood that there may be an initial treatment phaseduring which treatment is more frequent and/or in which higher doses areadministered. For example, in a subject with PNH or aHUS, it may requireseveral doses to achieve protection of a substantial fraction of thesubject's RBCs. After that, lower doses and/or less frequent dosingcould be used, e.g., to protect newly formed RBCs and/or to replenishprotection of existing RBCs. Of course similar approaches may befollowed for treatment of any disease where appropriate. In someembodiments treatment is started using IV administration and thenswitched to subcutaneous, intramuscular, or intradermal for maintenancetherapy. Depending on the disease, treatment may continue at intervalsfor, e.g., months, years, or indefinitely. Appropriate doses and dosingregimen depend at least in part upon the potency and half-life of thecompstatin analog (or other active agent), and may optionally betailored to the particular recipient, for example, throughadministration of increasing doses until a preselected desired responseis achieved, such as a desired degree of complement inhibition and/orcell protection. If desired, the specific dose level for any particularsubject may be selected based at least in part upon a variety of factorsincluding the activity of the specific compound employed, the particularcondition being treated, the age, body weight, general health, route ofadministration, the rate of excretion, any drug combination, and/or thedegree of complement protein expression or activity measured in one ormore samples obtained from the subject.

The invention encompasses administration of a compstatin analog incombination with additional therapy. Such additional therapy may includeadministration of any agent(s) used in the art or potentially useful fortreating a subject suffering from the disease.

When two or more therapies (e.g., compounds or compositions) are used oradministered “in combination” with each other, they may be given at thesame time, within overlapping time periods, or sequentially (e.g.,separated by up to 2 weeks in time), in various embodiments of theinvention. They may be administered via the same route or differentroutes. In some embodiments, the compounds or compositions areadministered within 48 hours of each other. In some embodiments, acompstatin analog can be given prior to or after administration of theadditional compound(s), e.g., sufficiently close in time that thecompstatin analog and additional compound(s) are present at usefullevels within the body at least once. In some embodiments, the compoundsor compositions are administered sufficiently close together in timesuch that no more than 90% of the earlier administered composition hasbeen metabolized to inactive metabolites or eliminated, e.g., excreted,from the body, at the time the second compound or composition isadministered.

In some embodiments, a composition that includes both the cell-reactivecompstatin analog and additional compound(s) is administered.

Example 1: Development of PEGylated Compstatin Analogs that RetainSubstantial Complement Inhibiting Activity

A compstatin analog having the amino acid sequence of the compstatinanalog of SEQ ID NO: 28, but incorporating an AEEAc-Lys moiety locatedC-terminal to the Thr residue of SEQ ID NO: 28 for purposes ofsubsequent conjugation of an NHS ester activated PEG to the amino groupof the Lys side chain was synthesized. The compound was synthesizedusing standard methods. Briefly, amino acids (including AEEAc) wereobtained as Fmoc-protected amino acids, in which the α-amino group ofeach amino acid was protected with Fmoc. Side chain functional groupswere also blocked with various appropriate protective groups. Synthesiswas accomplished following the solid phase methodology described byMerrifield (J. Amer. Chem. Soc. 85, 2149 (1963)). Chain assembly wasperformed on solid phase, at the conclusion of which the N-terminus wasacetylated; the peptide was then cleaved from the solid phase andsimultaneously deprotected via acidolysis using TFA and amidated. Thelinear peptide was then oxidized and purified. The resulting compstatinanalog is represented as follows

(SEQ ID NO: 51) Ac-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-AEEAc-Lys-NH2,abbreviated as CA28-AEEAc-Lys. Note that for purposes of brevity, theN-terminal acetyl group and C-terminal amino groups are omitted in thisabbreviation. Monofunctional, linear NHS-ester activated PEGs withmolecular weights of 30 kD and 40 kD (NOF America Corp. White Plains,N.Y., Cat. No. SUNBRIGHT® ME-300GS and Cat. No. SUNBRIGHT® ME-400GS),respectively, were coupled to the lysine side chain of CA28-AEEAc-Lys,resulting in long-acting compstatin analogs represented as follows:CA28-AEEAc-Lys-(PEG30k) and CA28-AEEAc-Lys-(PEG40k), and purified. Notethat the number after the term “PEG” and preceding the letter “k”represents the molecular weight of the PEG moiety in kilodaltons, andthe “k” is an abbreviation for kD). CA28-AEEAc-Lys-(PEG30k) is alsoreferred to as CA28-1. CA28-AEEAc-Lys-(PEG40k) is also referred to asCA28-2.

Inhibitory activity of the synthesized compounds was assessed bymeasuring the effect of the compounds on complement activation via theclassical pathway using a standard complement inhibition assay. Theprotocol measures C3b deposition in an ELISA format. C3b depositionmonitored using this method is generated through complement activated bythe classical pathway. Briefly, 96-well plates are coated with BSA.Human plasma, chicken ovalbumin (OVA), polyclonal anti-OVA antibodiesand compound being tested (referred to as “drug”) are added andincubated, followed by addition of Anti-human C3 HRP-conjugatedantibody. After an additional incubation, substrate is added and signaldetected. Details of the protocol are as follows:

Protocol for Classical Complement Inhibition Assay

Materials:

-   -   Ninety-six well plate (polystyrene plate, Thermo Scientific,        9205)    -   Chicken OVA (Sigma A5503-5G)    -   Rabbit anti-chicken OVA (Abcam ab1221)    -   Blocking buffer (Startingblock buffer, Thermo Scientific 37538)    -   Veronal Buffer (5× concentration, Lonza 12-624E)    -   Human plasma (collected with Lepirudin at 50 ug/ml final        concentration)    -   Goat anti-human C3 HRP-conjugated Ab (MP Biomedicals, 55237)    -   Tween-20 Wash Buffer (0.05% Tween 20-PBS buffer)    -   TMB (Peroxidase substrate, BD 555214)—1:1 mixture of BD        51-2607KC and 51-2606KC.    -   1M H₂SO₄        Protocol:

1. Add 100 ul/well of 1% chicken OVA (in PBS)

2. Incubate overnight @ 4° C. or room temperature for 1-2 hr.

3. Remove by shaking and tapping the plate.

4. Block by adding 200 ul of blocking buffer

5. Incubate for 1 h at room temp

6. Remove by shaking and tapping the plate

7. Add 100 ul of 1:1000 dilution of Polyclonal anti-chicken OVA inblocking buffer

8. Incubate for 1 h at room temp

9. Wash twice with wash buffer

10. Add 50 ul VB⁺⁺ to wells #2 to 12

11. Add 100 ul of starting drug dilution (2× in VB⁺⁺) to well 1.

12. Serially dilute (1:2) the drug from wells 1 to 10 as follow

-   -   a. Take 50 ul of solution from the originating well    -   b. Add this to the next well    -   c. Mix by pipetting several times    -   d. Repeat up to well #10

Note: from well #10 remove 50 ul and discard.

13. Add 50 ul of 2× plasma (1:37.5 dilution of original plasma) dilutionto wells 1 to 11

14. Incubate for 1 h

15. Wash with wash buffer

16. Add 100 ul of 1/1000 dilution of anti-C3-HRP Ab in blocking buffer

17. Incubate for 1 h

18. Wash with wash buffer

19. Add 100 ul of TMB to all wells

20. Incubate for 5-10 min in dark

21. Add 50 ul 1M H₂SO₄

22. Read the plate at 450 nm

VB⁺⁺

Formula:

Barbital   5 mM NaCl 72.5 mM MgCl₂  0.5 mM CaCl₂ 0.15 mM PH 7.4Stock Solutions:

Veronal Buffer (5X) Prod # MW For 500 ml 9 mM Sodium Barbitone SigmaB0500 206.17 927 mg 15.5 mM diethylbarbituric Sigma B0375 184.19 1.42grams acid Prod # MW For 50 ml Mg—Cl2 (200X) 100 mM MgCl₂—6H₂O SigmaM0250 203.30 1.00 gram CaCl₂ (500x) 75 mM CaCl₂ Sigma C7902 147.01551.28 mgTo prepare 50 ml of working buffer:

-   -   Weight 210 mg NaCl    -   Add 10 ml of 5× VB    -   Add 100 ul of CaCl₂ (500×)    -   Add 250 ul MgCl (200×)    -   Adjust volume to 50 ml with H₂O    -   Adjust pH to 7.4

Data was analyzed using GraphPad Prism5 software. Data sets from eachexperiment were normalized to percent activation compared to the 100%activation control corresponding to the well to which no compound isadded. Drug concentration values (X values) were transformed to theirlogarithms, and percent activation (Pa) (Y values) was transformed topercent inhibition (Pi) using the following formula Pi=100−Pa(Yi=100−Ya). The percent inhibition was plotted against the drugconcentration and the resulting data set was fit to a sigmoidal-doseresponse function [Y=Bottom+(Top−Bottom)/(1+10 ((Log EC−X)))]. IC₅₀values were obtained from the fit parameters.

Results are presented in FIG. 1, and the IC₅₀ values are shown in Table2 (in Example 2). As indicated, CA28-1 and CA28-2 displayed about 30% ofthe activity of CA28 on a molar basis.

Example 2: Development of Long-Acting Compstatin Analogs thatDemonstrate Increased Molar Activity

Eight-arm NHS-ester activated PEG with molecular weight of 40 kD (NOFAmerica Corp. White Plains, N.Y., Cat. No. SUNBRIGHT® HGEO-400GS;chemical formula: hexaglycerol octa(succinimidyloxyglutaryl)polyoxyethylene) was coupled to the lysine side chain of CA28-AEEAc-Lys,resulting in long-acting compstatin analogs represented as follows:(CA28-AEEAc)₈-PEG40k, also referred to as CA28-3. Complement inhibitingactivity of CA28-3 was tested using the assay described in Example 1.Results are plotted in FIG. 1 and IC50 value is listed in Table 3, bothas a function of CA28 concentration. The concentration of CA28 wascalculated using the extinction coefficient of CA28 at 283 nm (10208.14L·mol-1·cm-1). Based on other analysis (UV absorption vs. mass ofmaterial, and elemental CHN % analysis) it was concluded that there are7.5 CA28 moieties per molecule of CA28-3. Thus, the activity of CA28-3on a molar basis is 7.5-fold higher than shown in FIG. 1 and Table 3.Thus, the IC50 value in Table 3 is 7.5-fold higher than the actual IC50of CA28-3 on a molar basis. The IC50 of CA28-3 on a molar basis iscalculated as about 0.26 (lower than that of the parent compound CA28).FIG. 2 shows percent complement activation inhibiting activity of CA28and long-acting compstatin analogs CA28-2 and CA28-3, as a function ofCA28-3 concentration (μM), i.e., the activity of CA28-3 has beencorrected to account for the fact that the compound contains 7.5 CA28moieties. On a molar basis, the complement inhibiting activity of CA28-3exceeds that of CA28.

TABLE 3 CA28 CA28-1 CA28-2 CA28-3 IC50 0.3909 1.264 1.288 1.927

The solubility of CA28-1, CD28-2, and CA28-3 in water with or without avariety of buffer substances and/or excipients was observed to exceedthat of the parent compound CA28.

Example 3: Long-Acting Compstatin Analogs that Demonstrate DramaticallyIncreased Plasma Half-Life and Cmax

This Example describes determination of pharmacokinetic parameters oflong-acting compstatin analogs CA28-2 and CA28-3 followingadministration to Cynomolgus monkeys.

Dosing and Sample Collection

CA28-2 and CA28-3 were administered at time 0 via intravenous injectioninto female Cynomolgus monkeys (three per group, 2-5 yrs old, 2.9-3.5kg). Compounds were administered at 50 mg/kg in 5% dextrose in water ata concentration of 25 mg/ml. Blood specimens (˜1 mL each) were collectedfrom the femoral vein at the following timepoints: Pre-dose, 5 min, 15min, 30 min, 1 hour (h), 4 h, 8 h, 24 h, 48 h, 96 h (4 days), and 192 h(8 days) post dose. Specimens were collected via direct venipuncture andplaced into a red top serum tube containing no anticoagulant, and keptat room temperature for at least 30 minutes. Blood samples werecentrifuged at a temperature of 4° C. at 3000×g for 5 minutes. Sampleswere maintained chilled throughout processing. Serum samples werecollected after centrifugation and placed into sample tubes. Sampleswere stored in a freezer set to maintain −60° C. to −80° C. All animalsshowed normal activity throughout the study. No compound-relatedabnormalities were noted in the animals throughout the study.

Sample Analysis.

Plasma samples obtained as described above were analyzed by LC/MS/MSusing the following methods to determine the concentration of compound:50 μL of sample was mixed with internal standard (CA28-AEEAc-Arg) andthen 100 μL of 1 M NH₄OAc, pH 3.5 with HOAc was added and mixed. Then250 μL of acetonitrile was added and mixed. The sample was centrifugedand supernatant poured into another tube and dried. The sample wasreconstituted and injected onto the LC/MS/MS system. Mobile phase A was5 mM NH₄OAc with 0.1% FA and Mobile Phase B was 90:10 (ACN:50 mM NH₄OAc)with 0.1% FA. The LC column was the Intrada WP-RP 2×150 mm, 3μ.Quantitation was on an Applied Biosystems API-4000 triple quadrupolemass spectrometer operated in positive ion mode. In-source collisioninduced dissociation (CID) was used to fragment the compound in the massspectrometer source and the m/z 144 ion was mass selected in Q1,fragmented, and the m/z 77 ion mass selected in Q3 and detected. Datawas processed using Analyst 1.4.2 software.

Results.

The serum concentrations in micrograms/ml of CA28-2 and CA28-3 at eachtime point are presented in Table 4 below. Data for each of 3 monkeysthat received the indicated compound are shown. Average values andstandard deviations are readily calculated. There was notableconsistency between animals. CA28 are historical data obtained in aprevious study in which CA28 was administered intravenously toCynomolgus monkeys. In that study, CA28 was detected in samples usingHPLC.

TABLE 4 Serum Concentration in ug/mL CA28-3 CA28 CA28-2 Time (8-arm 40kPEG) (200 (linear 40k PEG) (days) (50 mg/kg) mg/kg) (50 mg/kg) 0.00351600 1330 1300 1460 1660 1610 0.01 1600 1220 1480 1360 1430 1530 0.021510 1170 1270 34 1310 1510 1500 0.042 1270 1030 1220 17 1290 1340 15400.167 926 893 934 9 1200 1210 1390 0.333 797 714 792 5 1190 1180 1 621479 558 1.5 927 853 881 2 384 355 360 612 733 760 4 280 252 262 461 458424 8 151 136 136 268 282 293

Results for each compound were averaged and are plotted in FIG. 3. Aremarkable increase in half-life and Cmax was observed for both CA28-2and CA28-3 compared to CA28. The terminal half-lives of both CA28-2 andCA28-3 were around 4-4.5 days. Based on these data, it is expected thatintravenous administration at approximately 1-2 week dosing intervalswill provide sustained levels of compound and effectively inhibitcomplement activation in human subjects, though shorter or longer dosingintervals may be used.

Example 4: Long-Acting Compstatin Analog Comprising HSA as a ClearanceReducing Moiety

Side chain lysines of human serum albumin (HSA) were converted to thiolsusing 2-iminothiolane and reacted with a compstatin analog comprising amaleimide as a reactive functional group:

(SEQ ID NO: 68) Ac-Ile-Cys*-Val-Trp(1-Me)-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr- AEEAc-Lys-(C(═O)—(CH₂)₅-Mal)-NH₂.The resulting long-acting compstatin analog (CA28-4) was tested in vitrofor complement inhibiting activity (FIG. 4) as described in Example 1and in vivo for pharmacokinetic properties as described in Example 3.Pharmacokinetic parameters of CA28-4 following administration toCynomolgus monkeys were determined as described in preceding example.Results are shown in FIG. 5 (along with results for CA28, CA28-1,CA28-2, and CA28-3). PK data for CA28-4 are presented in Table 5.

TABLE 5 Serum Concentration in ug/mL Time (hr) 0.0035 1790.0 1445.001395.00 0.0100 1195.0 915.50 885.00 0.0200 900.0 504.50 553.50 0.0420449.0 267.50 295.00 0.1670 194.0 164.00 158.50 0.3330 150.0 163.00119.50 1.0000 97.2 86.00 78.05 2.0000 73.3 51.55 57.40 4.0000 43.1 29.2034.15 8.0000 24.1 16.25 20.00

Example 5: Synthesis and Activity of PEG-Based Compstatin Analogs UsingDifferent NHS-Activated PEGs

A compstatin analog having the amino acid sequence of the compstatinanalog of SEQ ID NO: 28, but incorporating an AEEAc-Lys moiety locatedC-terminal to the Thr residue of SEQ ID NO: 28 for purposes ofsubsequent conjugation of an NHS ester activated PEG to the amino groupof the Lys side chain was synthesized as described in Example 1. Theresulting compstatin analog is represented as follows

(SEQ ID NO: 51) Ac-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-AEEAc-Lys-NH₂,abbreviated as CA28-AEEAc-Lys. Monofunctional, linearmonomethoxy-NHS-activated ester/carbonate PEGs with molecular weights of40 kD and differing in terms of the NHS carboxylate attachment chemistry(NOF America Corp. White Plains, N.Y., Cat. Nos. SUNBRIGHT® ME-400CS,SUNBRIGHT® ME-400GS, SUNBRIGHT® ME-400HS, SUNBRIGHT® ME-400TS) werecoupled to the lysine side chain of CA28-AEEAc-Lys via an amide bond.(The Lys reside is Lys15 since the AEEAc linker contains an amino acidresidue.) All compounds were acetylated on the N-terminus, amidated onthe C-terminus, and cyclized via a disulfide bond between Cys2 andCys12. (The acetylation, amidation, and cyclization were performed priorto coupling to the PEG.) The compounds were prepared as trifluoroacetatesalts and were purified. The compounds are represented as shown in thefollowing table (Table 6). The letters CS, GS, HS, and TS represent thedifferent linker moieties between the PEG moiety and the NHS moiety asindicated in further detail in Table 6. It will be understood thatvarious names and abbreviations for each compound may be usedinterchangeably. Note that CA28-2 (see Example 1) is the same asCA28-2GS.

TABLE 6 PEG-Based Compstatin Analogs Containing One Compstatin AnalogMoiety Compound Abbreviation and ID Compound Name*^(,†) Activated PEGCA28-2CS CA28-AEEAc-LysCS = PEG40K: Methoxy-PEG-CO(CH₂)₂COO—NHS (NOFCA28-AEEAc- Sunbright 400CS) Lys(mPEG40K-succinyl) Chemical Name:α-Succinimidyloxysuccinyl-ω-methoxy, polyoxyethylene CAS#: 78274-32-5CA28-2GS CA28-AEEAc-LysGS PEG40K: Methoxy-PEG-CO(CH₂)₃COO—NHS (NOF (alsoreferred CA28-AEEAc- Sunbright 400GS) to as CA28-2) Lys(mPEG40K-Chemical Name: α-Succinimidyloxyglutaryl-ω-methoxy, pentanedioyl)polyoxyethylene CAS#: 111575-54-3 CA28-2HS CA28-AEEAc-LysHS PEG40K:Methoxy-PEG-(CH₂)₅COO—NHS (NOF CA28-AEEAc- Sunbright 400HS)Lys(mPEG40K-hexanoyl) Chemical Name: Poly(oxy-1,2-ethanediyl),α-methyl-ω- {2-[(2,5-dioxo-1-pyrrolidinyl)oxy]-6-oxohexyloxy}- CA28-2TSCA28-AEEAc-LysTS PEG40K: Methoxy-PEG-COO—NHS (NOF Sunbright CA28-AEEAc-400TS) Lys(mPEG40K-carbonyl) Chemical Name: α-Succinimidylcarbonyl-ω-methoxy, polyoxyethylene CAS# 135649-01-3 *AEEAc = 8 =Amino-3,6-dioxa-octanoyl ^(†)Compounds were prepared as trifluoroacetatesalts but other counterions could be used

Compounds were analyzed by reverse phase HPLC. FIG. 6 shows arepresentative chromatogram for one of the compounds. A VariTide RPCcolumn was used. Eluent A was 0.1% TFA in water; Eluent B was 0.1% TFAin 50% CAN/40% water. Flow rate was 1.000 ml/min with a gradient of 0% Bto 100% B over 40 minutes. The peak with a retention time of 33.68minutes represents the PEGylated compound and has a relative area of96.50%.

Inhibitory activity of the compounds was assessed by measuring theeffect of the compounds on complement activation via the classicalpathway using a standard complement inhibition assay as described inExample 1. Results are plotted in FIG. 7. These results represent acombination of two separate experiments. The compounds showed notablysimilar complement inhibiting activity.

Example 6: Synthesis and Activity of Bifunctionalized PEG-BasedCompstatin Analogs

Bifunctional, linear monomethoxy-NHS-activated ester/carbonate PEGs withmolecular weights of 40 kD and differing in terms of the NHS carboxylateattachment chemistry were obtained from NOF America Corp. (White Plains,N.Y.). The activated PEGs were coupled to the lysine side chain ofCA28-AEEAc-Lys via an amide bond such that two CA28-AEEAc-Lys moietieswere coupled to each PEG chain. All compounds were acetylated on theN-terminus and amidated on the C-terminus of the CA28-AEEAc-Lysmoieties, and cyclized via a disulfide bond between Cys2 and Cys12. (Theacetylation, amidation, and cyclization were performed prior to couplingto the PEG.) The compounds were prepared as acetate salts and werepurified. The compounds are represented as shown in the following table(Table 7).

TABLE 7 Bifunctionalized PEG-Based Compstatin Analogs CompoundAbbreviation and ID Compound Name*^(,†) Activated PEG CA28-2CS-BFCA28-AEEAc-LysCS = PEG40K: NHS—OCO(CH₂)₂COO-PEG-CO(CH₂)₂COO—NHSCA28-AEEAc- Chemical Name: α-Succinimidyloxysuccinyl-ω-Lys(mPEG40K-succinyl) succinimidyloxysuccinyloxy, polyoxyethylene CAS#:85419-94-9 CA28-2GS-BF CA28-AEEAc-LysGS PEG40K:NHS—OCO(CH₂)₃COO-PEG-CO(CH₂)₃COO—NHS CA28-AEEAc- Chemical Name:α-Succinimidyloxyglutaryl-ω- Lys(mPEG40K- succinimidyloxyglutaryloxy-,polyoxyethylene pentanedioyl) CAS#: 154467-38-6 CA28-2HS-BFCA28-AEEAc-LysHS PEG40K: NHS—OCO(CH₂)₅O-PEG-(CH₂)₅COO—NHS CA28-AEEAc-Chemical Name: α-[6-[(2,5-dioxo-1-pyrrolidinyl)oxy]-6-Lys(mPEG40K-hexanoyl) oxohexyl]-ω-[6-[(2,5-dioxo-1-pyrrolidinyl)oxy]-6-oxohexyloxy]-, polyoxyethylene CA28-2TS-BF CA28-AEEAc-LysTS PEG40K:NHS—OCO-PEG-COO—NHS CA28-AEEAc- Chemical Name: α-Succinimidylcarbonyl-ω- Lys(mPEG40K-carbonyl) Succinimidyl carbonyl, polyoxyethylene*AEEAc = 8 = Amino-3,6-dioxa-octanoyl ^(†)Compounds were prepared asacetate salts but other counterions could be used

Inhibitory activity of CA28-2GS-BF was assessed by measuring the effectof the compound on complement activation via the classical pathway usinga standard complement inhibition assay as described in Example 1 andanalyzed as described in Example 1. Results are plotted in FIG. 8. Asdescribed above, CA28-2GS-BF contains two compstatin analog moieties permolecule. Although the activity per compstatin analog moiety ofCA28-2GS-BF is less than the activity of an individual CA28 molecule,the activity of the two compounds on a molar basis is virtuallyidentical over a broad range of concentrations.

Example 7: Subcutaneous Administration of Bifunctionalized PEG-BasedCompstatin Analog

This Example describes determination of pharmacokinetic parameters oflong-acting compstatin analog CA28-2GS-BF following administration toCynomolgus monkeys either via a single intravenous (IV) injection orwith repeated (once daily) subcutaneous administration for seven days.

Dosing and Sample Collection

CA28-2GS was administered at time 0 via intravenous injection or viarepeat subcutaneous injection (daily, for seven days) into maleCynomolgus monkeys. Six non-naïve male Cynomolgus monkeys, age 1-5years, ranging in weight from 4.6 to 5.3 kilograms, were used in thestudy (three per group). The animals were healthy at the start of thetrial seven days. The study was not blinded. Animals were supplied withwater ad libitum and a commercial diet twice daily prior to initiationof the study. Food was supplied to the animals per facility SOP prior tothe study. Animals were not fasted. Animals were dosed via intravenousand subcutaneous administration at time 0 on the appropriate day. A size22 gauge needle was used for the subcutaneous administration. Thecompound was administered at 50 mg/kg in 5% dextrose in water at aconcentration of 25 mg/ml. Blood specimens (˜1 mL each) were collectedfrom the femoral vein at the following timepoints: Day 1: Pre-dose, 5min, 15 min, 30 min, 1 hour (h), 4 h, 8 h. Days 2-9: 0 min. Day 16:Final sample based on Day 1 dosing. Each blood sample (˜1.0 mL) wascollected from the monkey's femoral or saphenous vein via directvenipuncture and placed into a red top serum tube containing noanticoagulant, and kept at room temperature for at least 30 minutes.Blood samples were centrifuged at a temperature of 4° C. at 3000×g for 5minutes. Samples were maintained chilled throughout processing. Serumsamples were collected after centrifugation and placed into sampletubes. Samples were stored in a freezer set to maintain −60° C. to −80°C. Serum samples and leftover dosing solutions were shipped frozen ondry ice for analysis.

The site of each subcutaneous administration was observed to see howfast the injection volume was absorbed and also to see if theformulation left behind a lump or fully went away. The dose sites wereobserved at each collection timepoint and in the afternoon of days 2-7.All doses were absorbed during the duration of the study. Based on theobservations it is estimated that doses were absorbed within fifteenminutes after administration. All animals showed normal activitythroughout the study. No compound-related abnormalities were noted inthe animals throughout the study.

Sample Analysis.

Plasma samples obtained as described above were analyzed by LC/MS/MSleveraging CID (collision induced degradation) similarly to the methoddescribed in Example 3.

Results.

Serum concentration vs time for CA28-2GS-BF when administered IV orsubcutaneously as described above are plotted in FIG. 9, The data pointsrepresent all PEGylated CA28 compound detected. CA28 data shown on FIG.9 are historical data obtained in a previous study in which CA28 wasadministered intravenously to Cynomolgus monkeys. In that study, CA28was detected in samples using HPLC.

A peak serum concentration of 500 μg/ml (11 μM) was achieved bysubcutaneous administration of CA28-2GS-BF. The terminal half-life ofCA28-2GS-BF was approximately 5 days when administered either IV orsubcutaneously. Results are summarized in the tables below:

TABLE 8 Summary of Study Sample Concentrations for CA28-2GS-BF in MonkeySerum (IV @ 50 mg/kg on Day 0) CA28-2GS-BF Conc. (μg/mL) TimepointAnimal 1 Animal 2 Animal 3  5 min 1850 1550 2030 15 min 1760 1440 200030 min 1560 1380 1810  1 hr 1650 1330 1710  4 hr 1270 1000 1510  8 hr1050 913 1360 Day 2 684 661 711 Day 3 541 471 538 Day 4 463 417 492 Day5 366 384 389 Day 6 346 331 358 Day 7 303 306 311 Day 8 257 252 259 Day9 217 252 233 Day 15 92.8 107 95.6

TABLE 9 Summary of Study Sample Concentrations for CA28-2GS-BF in MonkeySerum (SQ @ 7 mg/kg/day × 7 days) CA28-2GS-BF Conc. (μg/mL) TimepointAnimal 1 Animal 2 Animal 3  5 min 0 5.18 3.55 15 min 0 5.57 3.47 30 min0 4.66 3.93  1 hr 0 5.41 3.56  4 hr 4.41 12.6 11.1  8 hr 32.0 15.3 21.5Day 2 54.2 56.6 53.5 Day 3 135 117 122 Day 4 248 260 234 Day 5 398 316311 Day 6 447 391 419 Day 7 564 412 448 Day 8 591 432 468 Day 9 596 423455 Day 15 152 241 199

Example 8: Inhibition of Complement-Mediated Lysis of Red Blood Cellsfrom Patients with PNH

A modified Ham's test is performed to measure the ability of compstatinanalogs to inhibit complement-mediated lysis of red blood cells frompatients with PNH in vitro. Complement is activated by acidified serumwith added magnesium to lyse the PNH red cells. The incubation isperformed for 90 minutes. The read out is flow cytometry for PNH redcells using standard markers. Heat inactivated serum is used as acontrol (produces no hemolysis). Acidified serum in the absence of addedcomplement inhibitor produces maximum lysis. The experiment is performedwith serial two-fold dilutions of compstatin analogs CA28, CA28-2,CA28-2CS, CA28-2CS-BD, CA28-2GS, CA28-2GS-BF, CA28-2HS, CA28-2HS-BF,CA28-2TS, CA28-2GS-BF, and CA28-3. The concentration of each compoundrequired to fully block hemolysis in vitro is determined. Red bloodcells are also stained for C3 fragment deposition using anti-C3polyclonal antibodies that do not contain any bridge leading toagglutination (e.g., either Ab4214 or Ab14396, both commerciallyavailable FITC-conjugated Abcam, Cambridge, United Kingdom) in order tomeasure the ability of the compounds to inhibit deposition of C3fragments on PNH red blood cells. Results are compared with thoseobtained with eculizumab using the same assays.

Example 9: Long-Acting Compstatin Analogs in Patients with PNH

A cohort of subjects diagnosed with PNH is divided into 4 groups.Subjects in Groups 1 and 2 are treated with intravenous administrationof CA28-2 or CA28-3, respectively, at a dose of between 5 mg/kg and 20mg/kg, at time intervals between 1 and 2 weeks. Optionally, treatment isstarted at more frequent time intervals and then reduced in frequencyfor maintenance therapy. Subjects in Group 3 are treated with eculizumabaccording to the recommended dosing regimen. Group 4 serves as a control(no complement inhibitor therapy). Intravascular hemolysis (based on LDHmeasurement and/or (51)Cr labeling of RBCs), reticulocytosis (anindicator of anemia), hematocrit, hemoglobin concentration in the blood,opsonization of red blood cells (deposition of products of C3activation, such as C3b, on red blood cells, which may be detected usingflow cytometry), PNH symptoms, transfusion requirements, thromboembolicevents, haemolysis-associated nitric oxide depletion, measures ofpulmonary hypertension, quality of life, and survival are monitored overtime. Results are compared between groups and with historical data fromcontrol PNH patients obtained in clinical trials of eculizumab. Animprovement in persistent anemia (e.g., as evidenced by reducedreticulocytosis, reduced evidence of hemolysis, increased hematocrit,increased hemoglobin), improved quality of life, reduced PNH symptoms,reduced transfusion requirements, reduced thromboembolic events, reducedhaemolysis-associated nitric oxide depletion, reduced measures ofpulmonary hypertension increased quality of life, and/or increasedsurvival, in subjects receiving CA28-2 (Group 1) or CA28-3 (Group 2), ascompared with subjects in Group 4 is indicative of efficacy.

Example 10: Long-Acting Compstatin Analogs in Patients with PNH

Example 9 is repeated with the modification that subjects areindividuals with PNH who remain transfusion-dependent and/or continue tohave a hemoglobin below a cutoff (such as 9.0 g/dL) despite treatmentwith eculizumab. Results are compared among groups.

Example 11: Long-Acting Compstatin Analogs in Patients with aHUS

A cohort of subjects diagnosed with aHUS is divided into 4 groups.Subjects in groups 1 and 2 are treated with intravenous administrationof CA28-2 or CA28-3, respectively, at a dose of between 5 mg/kg and 20mg/kg, at time intervals between 1 and 2 weeks. Optionally, treatment isstarted at more frequent time intervals and then reduced in frequencyfor maintenance therapy. Subjects in Group 3 are treated with eculizumabaccording to the recommended dosing regimen. Intravascular hemolysis(based on LDH measurement), opsonization of red blood cells (depositionof products of C3 activation, such as C3b, on red blood cells), aHUSsymptoms, renal function, need for plasma exchange or dialysis, qualityof life, and survival are monitored over time. Results are comparedbetween groups and with historical data from control aHUS patientsobtained in clinical trials of eculizumab. Reduced evidence ofhemolysis, improved quality of life, reduced aHUS symptoms, reduced needfor plasma exchange or dialysis, increased quality of life, and/orincreased survival, in subjects receiving CA28-2 or CA28-3, as comparedwith subjects in group 4 are indicative of efficacy.

Example 12

Examples 8-11 are repeated using CA28-2GS-BF, CA28-2HS, CA28-2HS-BF,CA28-2TS, and CA28-2GS-TS-BF.

Example 14

Examples 9-12 are repeated using CA28-2GS-BF, CA28-2HS, CA28-2HS-BF,CA28-2TS, and CA28-2GS-TS-BF administered daily by subcutaneousinjection.

Example 14

Examples 8-11 are repeated using additional long-acting compstatinanalogs.

Example 15

Examples 8-11 are repeated using cell-reactive compstatin analogs.

Example 16: Complement Activation Inhibiting Activity of a Long-ActingCompstatin Analog

CA28 and CA28-AEEAc-Lys were synthesized as described above. CA28-2TS-BFwas synthesized using a reactive bifunctional PEG of the TS type interms of the NHS carboxylate attachment chemistry, which was linked totwo molecules of CA28-AEEAc-Lys via the primary amine of the lysine sidechain. The complement activation inhibitory activity of CA28 andCA28-2TS-BF was assessed by measuring the effect of the compounds oncomplement activation via the classical pathway and via the alternativepathways using standard complement inhibition assays. The protocol forthe classical pathway activation assay is described in Example 1. Theprotocol for alternative pathway activation also measures C3b depositionin an ELISA format and is described below. C3b deposition monitoredusing this method is generated through complement activated by thealternative pathway by lipopolysaccharide (LPS). Briefly, 96-well platesare coated with LPS. Compound being tested (referred to as “drug”) isadded, followed by addition of plasma or serum as a source ofcomplement, and incubated. This is followed by addition of anti-human C3HRP-conjugated antibody. After an additional incubation, substrate isadded and signal detected. Details of the protocol are as follows:

ELISA-Based Assay for Alternative Complement Pathway Activation

Materials:

-   -   Ninety six-well ELISA plate (Corning 3590)    -   LPS from Salmonella typhosa—Sigma L7136 (40 ug/ml in PBS)    -   BSA 1% in PBS—Calbiochem #126626 1/30 dilution    -   Veronal Buffer+10 mM MgCl₂+10 mM EGTA (VB-Mg EGTA)    -   Human plasma (collected with Lepirudin at 5 ug/ml final        concentration)    -   Anti-human C3 HRP-conjugated Ab (Poli to C3-HRP Ab, Cappel        55237)    -   Tween-20 Wash Buffer (0.05% in PBS)    -   TMB (Peroxidase substrate)—1:1 mixture of BD 51-2607KC and        51-2606KC.    -   3M H₂SO₄    -   Micro-plate Reader        Protocol:

1. Add 50 ul/well of LPS at 40 ug/ml (in PBS)

2. Incubate for 2 hours at room temp

3. Remove by shaking and tapping the plate.

4. Block by adding 200 ul of 1% BSA/PBS

5. Incubate for 1 h at room temp

6. Remove by shaking and tapping the plate

7. Add 50 ul VB-Mg EGTA to wells #2 to 12

8. Add 100 ul of starting drug dilution (2× in VB-Mg EGTA) to well 1.

9. Serially dilute (1:2) the drug from wells 1 to 10 as follow

-   -   a. Take 50 ul of solution from the originating well    -   b. Add this to the next well    -   c. Mix by pipetting several times    -   d. Repeat up to well #10

Note: from well #10 remove 50 ul and discard.

10. Add 50 ul of 2× plasma dilution to wells 1 to 11

11. Incubate for 1 h

12. Wash twice with wash buffer

13. Add 50 ul of 1/1000 dilution of C3-HRP Ab in 1% BSA/PBS

14. Incubate for 1 h

15. Add 100 ul of TMB to all wells

16. Incubate for 30 min

17. Add 50 ul 3M H₂SO₄

18. Read the plate at 450 nm

Formula for VB Mg EGTA

Barbital  5 mM NaC1 72.5 mM   MgC12 10 mM EGTA 10 mM pH 7.3-7.4Stock Solutions:

Veronal Buffer (5X) Prod # MW For 500 ml 9 mM Sodium Barbitone SigmaB0500 206.17 927 mg 15.5 mM diethylbarbituric Sigma B0375 184.19 1.42grams acid Mg—Cl2 (10X) Prod # MW For 50 ml 100 mM MgCl₂—6H₂O SigmaM0250 203.30 1.00 gram EGTA (10x) Prod # MW For 25 ml 100 mM EGTA SigmaE8145 468.3 1.17 gramsTo Prepare 20 ml of Working Buffer:

-   -   Weight 84 mg NaCl    -   Add 4 ml of 5×VB    -   Add 2 ml of EDTA 10×    -   Add 2 ml MgCl 10×    -   Adjust volume to 20 ml with H₂O    -   Adjust pH to 7.4

Results

FIG. 10(A) shows percent inhibition of classical complement activationinhibiting activity by CA28 and CA28-2TS-BF as a function of molarconcentration of the compounds. FIG. 10(B) shows percent inhibition ofalternative complement activation inhibiting activity by CA28 andCA28-2TS-BF as a function of molar concentration of the compounds. Rawdata are tabulated in Table 10 below (4 replicates of each condition).Based on the inhibition curves shown in the figures and underlying data,the complement inhibiting activity of CA28-2TS-BF is at least as greatas that of CA28 on a molar basis within the experimental error of theassay. These results further confirm the suitability of long-actingcompstatin analogs described herein, e.g., for therapeutic purposes.

TABLE 10 % inhibition Conc uM CA28 CA28-2TS-BF AP Inhibition 25 90.0790889.9353 90.65421 89.21639 90.25768 91.10484 90.68126 90.96365 12.588.92883 89.21639 89.21639 90.94177 90.11649 89.12814 89.26933 88.563366.25 87.05967 89.07261 88.20992 88.64127 88.28098 87.29263 87.4338287.43382 3.125 85.62186 87.49101 87.49101 86.77211 84.04518 84.4687785.17473 85.03353 1.5625 70.81236 81.30841 82.02732 70.38102 77.4091169.5023 79.80939 81.08012 0.78125 58.01582 55.57153 57.15313 61.7366866.39605 72.74974 61.3131 0.390625 18.04458 12.4371 23.65205 24.9460856.79492 44.9347 56.65372 40.59891 0.1953 15.02516 15.31273 15.3127322.34381 23.89693 26.01483 20.6495 0.09765 2.803741 14.30625 6.6858445.823158 13.87222 6.953766 10.62479 0.3176955 0.0488 −1.365921 5.6793670.790802 −2.803734 0.6000748 2.012009 0.3176956 −2.929749 0 −2.803734−1.509697 1.078362 3.235085 0.6000748 7.094963 2.153198 −0.5294724 CPInhibition 25 83.69539 79.90365 81.00482 82.09877 81.32716 77.31482 12.578.66483 67.24019 81.69305 83.75774 83.48766 80.70988 78.54939 76.080256.25 84.58362 84.03304 80.45423 81.28011 81.94444 78.39507 79.9382774.84568 3.125 83.62009 81.69305 79.90365 81.8307 79.93827 77.932177.46913 76.69753 1.5625 76.04955 76.60014 79.76601 78.94012 71.6049370.52469 74.53703 75.61728 0.78125 71.50723 69.85547 73.98486 72.3331172.37654 68.20988 72.0679 71.14198 0.390625 58.84377 72.05782 68.8919563.11081 79.16666 70.37037 71.2963 62.19136 0.1953 42.60152 44.3909166.51234 47.68519 50.92593 0.09766 24.7075 24.15692 23.05575 35.0309841.66657 35.03086 48.91975 42.12963 0.0488 15.7605 12.59464 15.2099227.04749 12.50001 26.23457 26.23457 23.61111 0 −20.99105 7.08878312.04405 1.858231 5.09259 −0.1543198 −0.9259262 −4.012352

Example 17: Pharmacokinetic Properties of Long-Acting Compstatin AnalogAdministered by the Intravenous or Subcutaneous Route

This Example describes determination of pharmacokinetic parameters oflong-acting compstatin analog CA28-2TS-BF following administration toCynomolgus monkeys with a single intravenous (IV) injection, singlesubcutaneous administration, or with once daily subcutaneousadministration for seven days. CA28-2TS-BF was synthesized using areactive bifunctional PEG of the TS type in terms of the NHS carboxylateattachment chemistry, which was linked to two molecules ofCA28-AEEAc-Lys via the primary amine of the lysine side chain.

Dosing and Sample Collection

CA28-2TS-BF was administered to Cynomolgous monkeys at time 0 viaintravenous injection into the saphenous vein or via single subcutaneousinjection or repeat subcutaneous injection (once daily, for seven days).Six non-naïve female Cynomolgus monkeys, age 2-5 years, ranging inweight from 2.6 to 3.9 kilograms, were used in this study (three pergroup). The animals were healthy at the start of the trial. The studywas not blinded. Animals were supplied with water ad libitum and acommercial diet twice daily prior to initiation of the study. Food wassupplied to the animals per facility SOP prior to the study. Animalswere not fasted. Animals were dosed at 7 mg/kg via intravenous orsubcutaneous administration at time 0 on the appropriate day(s). Dosingsolution concentration was 3.5 mg/mL for IV administration and 25 mg/mLfor subcutaneous administration. Dosing volume was 2 mL/kg for IVadministration and 0.28 mL/kg for subcutaneous administration. A size23G3/4 gauge needle was used for subcutaneous administration. Thecompound was administered in 5% dextrose in water.

Blood specimens (˜0.5-1 mL) were collected from the femoral vein at thefollowing timepoints: Day 1: Pre-dose, 5 min, 15 min, 30 min, 1 hour(h), 4 h, 8 h. Days 2-9: 0 min. Day 15: Final sample based on Day 1dosing. Each blood sample was collected from the monkey's femoral veinvia direct venipuncture and placed into a red top serum tube containingno anticoagulant, and kept at room temperature for at least 30 minutes.Blood samples were centrifuged at a temperature of 4° C. at 3000×g for 5minutes. Samples were maintained chilled throughout processing. Serumsamples were collected after centrifugation and placed into sampletubes. Samples were stored in a freezer set to maintain −60° C. to −80°C. Serum samples and leftover dosing solutions were shipped frozen ondry ice for analysis.

The site of each subcutaneous administration was observed to see howfast the injection volume was absorbed and also to see if theformulation left behind a lump or fully went away. The dose sites forthe animals receiving subcutaneous injections were observed in theevening of each dosing day. The dose site did not appear to have a lumpand was fully absorbed by that time based on visual inspection. Allanimals were observed twice daily and showed normal activity throughoutthe study. No compound-related abnormalities were noted in the animalsthroughout the study.

Sample Analysis.

Plasma samples obtained as described above were analyzed by LC/MS/MSleveraging CID (collision induced degradation) similarly to the methoddescribed in Example 3.

Results.

Serum concentrations vs time for CA28-2TS-BF when administered IV orsubcutaneously as described above are plotted in FIG. 11. The datapoints represent all PEGylated CA28 compound detected. CA28 data shownon FIG. 11 are historical data obtained in a previous study in whichCA28 was administered intravenously to Cynomolgus monkeys. In thatstudy, CA28 was detected in samples using HPLC/MS.

A peak serum concentration of about 500 micrograms/mL was achieved bysubcutaneous administration of CA28-2TS-BF once daily for 7 days. Theterminal half-life of CA28-2TS-BF was approximately 8 days whenadministered either IV or by single subcutaneous injection. Raw data areprovided in Tables 11(A) (IV administration) and 11(B) (subcutaneousadministration) below. (In FIG. 11 and Tables 11(A) and 11(B), the dayof dosing is considered day 0).

TABLE 11(A) CA28-2TS-BF CA28 (IV) (single dose IV) Time (days) 200 mg/kg7 mg/kg 0.0035 232 190 214 0.01 216 190 209 0.02 34 221 177 199 0.042 17211 175 183 0.167 9 190 152 185 0.333 5 212 191 154 1 1.5 180 130 150 2141 116 126 3 128 98.7 113 4 114 89.1 95.8 5 105 75 87.1 6 95.1 67 74.47 83.3 61.4 69.9 8 86 52.8 68.4 14 51 30.8 39.7

TABLE 11(B) Time CA28-2TS-BF (single dose SC) CA28-2TS-BF (7 × daily SC)(days) 7 mg/kg 7 mg/kg/day 0.0035 BQL BQL BQL BQL BQL BQL 0.01 1.42 BQL1.7 BQL BQL BQL 0.02 3.55 1.64 3.8 BQL BQL BQL 0.042 6.1 3.46 7.05 2.321.14 2.44 0.167 15 12.2 20.6 14.7 5.76 12.1 0.333 25 25.8 32.9 32.9 1825.8 1 70 76.5 76 80.5 80 66.5 2 107 101 96.6 196 185 169 3 111 103 99.8391 286 292 4 108 98.9 99.5 455 377 405 5 99.4 97.6 101 427 404 486 686.8 87 81.6 490 483 568 7 75.2 83.2 78.6 607 502 564 8 67.5 73.4 72.2495 481 570 14 38.3 44.5 40.7 322 298 397 BQL = below quantificationlimit

As noted above, CA28-2TS-BF was synthesized using a reactivebifunctional PEG of the TS type, resulting in formation of a carbamateafter reaction with the primary amine of lysine. CA28-2GS-BF wassynthesized using a reactive bifunctional PEG of the GS type in terms ofthe NHS carboxylate attachment chemistry, resulting in formation of anamide after reaction with the primary amine of lysine. The compound alsocontains an ester linkage, which is absent in CA28-2TS-BF. It is notablethat the terminal half-life of about 8 days achieved with CA28-2TS-BF inthis experiment was considerably greater than that of CA28-2GS-BF, whichwas found to have a half-life of about 5 days in a similar experiment(see Example 8).

Example 18: Compstatin Analogs Inhibit C3 Deposition on Red Blood Cellsof PNH Patients and Protects Against Complement-Mediated Lysis

A modified Ham's test was performed to assess the ability of compstatinanalogs to protect PNH RBCs from complement-mediated lysis. RBCs from apatient with PNH were exposed to acidified human serum (as a source ofcomplement components) and magnesium (Mg²⁺, needed for alternativepathway activation) in the absence of complement inhibitors or in thepresence of varying amounts of compstatin analogs CA28 or CA28-2GS-BF.Exposure to heat inactivated human serum was used as a controlrepresenting no significant complement-mediated lysis as complement isinactivated by heat. Exposure to acidified human serum and magnesium(Mg²⁺) in the absence of complement inhibitors (pane labeled Mg²⁺) wasused as a control representing maximum lysis.

Following incubation, cells were stained with antibodies to CD59 andC3d. Cd59 level permitted the classification of the PNH RBCs as Type I,Type II, or Type III. Staining for C3d, a product of C3 activation andcleavage was used as a marker of C3 and C3 activation product deposition(loading). Flow cytometric analysis was performed to assess CD59 and C3don RBC surfaces and to quantify the percentages of Type I, Type II, andType III cells present in various samples.

Results of a dilution experiment demonstrating the effect of differentconcentrations of CA28 on C3 deposition and cell percentages are shownin FIG. 12(A). Results of a dilution experiment demonstrating the effectof different concentrations CA28-2GS-BF on C3 deposition and cellpercentages are shown in FIG. 12(B). The results are presentedquantitatively in Table 12 below. Type I cells (shown in orange in FIG.12) have normal levels of CD59. Type III cells (shown in blue in FIG.12) have essentially no detectable CD59. These cells are verysusceptible to complement-mediated lysis. Type II cells (shown in purplein FIG. 12) have reduced levels of CD59 as compared with normal or TypeI cells and have an intermediate sensitivity to complement-mediatedlysis. In the presence of complement activation Type III cells rapidlylyse. Reduction in or absence of lysis may be evidenced by an increasedpresence of Type III cells, as is evident as a higher percentage of TypeIII cells in the no lysis panel compared with the panel in (presence ofMg²⁺ (max. lysis)) in both FIGS. 12(A) and 12(B). In other words, thereare relatively fewer Type III cells in the positive control than in thenegative control. Type II cells may eventually lyse in the presence ofactivated complement but can accumulate a considerable amount of C3activation products such as C3d before they do so. Reduction in orabsence of lysis may be evidenced by increased levels of C3 or C3activation products on Type II cells, as is evident by comparing thelevel of C3d on Type II cells in the no lysis panel with the level ofC3d on Type II cells in the max. lysis panel in both FIGS. 12(A) and12(B). In other words, there is more C3d on cells in the max. lysispanel than in the no lysis panel. Type I cells have functional CD59, sothey deactivate convertase and therefore do not accumulate as much C3das Type II cells. However, the amount of C3d they accumulate can be usedas a surrogate indicator for the amount of lysis of the more vulnerablecells (Type II and III). Thus, reduced C3d on Type I cells is indicativeof protection against lysis. A shift in the relative percentages of TypeI, II, and III cells from the percentages present in the max. lysiscontrol panels (Mg²⁺) towards the percentages present in the no lysiscontrol panels (heat inactivated serum) is indicative of protectionagainst complement-mediated lysis. These percentages are shown in thetable below. The column labeled % C′3 in Table 12 refers to thepercentage of cells deemed “positive” for presence of C3 and C3activation products (“C3 loading”). As can be seen in FIGS. 12(A) and(B) and Table 12, CA28 and CA28-2GS-BF demonstrated similar protectionof PNH red cell lysis over the concentrations tested, with virtually noC3 loading on PNH red cells at 100 micrograms/ml compound or higherconcentrations. Note that the percentages of Type III, II, and I cellsin the presence of 100 ug/ml or more compstatin analog were essentiallythe same as in the no lysis control, indicating complete protection fromcomplement-mediated lysis as determined by this assay. Concentrationsbelow 100 ug/ml but above 60 ug/ml, e.g., at least 70 ug/ml, at least 80mg/ml, or at least 90 ug/ml, but below 100 ug/ml were not tested in thisexperiment but may also provide significant protection. 100micrograms/ml CA28-2GS-BF represents a concentration of about 2.5micromolar, which is readily achievable in vivo as described herein.

TABLE 12 Percentages and C3 loading of Type I, II, and III PNH RBCs inthe absence or presence of compstatin analogs (concentrations inmicrograms/ml are shown) Type % Type % Type % III % C′3 II % C′3 I % C′3Heat inactivated 37.09 0.29 51.79 1.22 11.12 0.09 (no lysis) Magnesiumadded 15.75 0.96 64.7 13.48 19.55 3.2 (maximum lysis) CA28 1 19.42 1.264.81 10.08 15.77 1.7 CA28 4 18.43 0.83 64.9 10.42 16.66 1.76 CA28 817.83 0.7 66 11.12 16.17 1.65 CA28 15 22.11 0.96 62.9 8.63 15 1.93 CA2825 20.53 0.75 64.51 9.3 14.96 1.84 CA28 100 37.96 0.1 51.5 0.92 10.640.08 CA28 500 37.29 0.09 52.06 0.83 10.64 0.14 CA28-2GS-BF 20 19.19 0.9965.35 12.28 15.46 2.18 CA28-2GS-BF 40 15.15 1.21 68.96 15.12 15.89 2.84CA28-2GS-BF 50 13.87 1.29 69.46 15.94 16.67 2.26 CA28-2GS-BF 60 17.941.44 66.97 13.04 15.09 2.05 CA28-2GS-BF 100 35.32 0.11 53.93 0.88 10.750.07 CA28-2GS-BF 200 37.43 0.02 51.99 0.26 10.59 0.02 CA28-2GS-BF 50037.87 0.05 51.87 0.3 10.26 0.04

Example 19: Effect of Compstatin Analog and Soliris on C3 Deposition onRed Blood Cells from PNH Patient

A similar experiment to that described in Example 18 was performed tofurther demonstrate the protective effect of compstatin analogCA28-2GS-BF and compare it with that of anti-C5 antibody Soliris. Amodified Ham's assay as in Example 18 was performed using PNH RBCsincubated in the presence of activated complement either in the absenceof complement inhibitor (left panel) or in the presence of Soliris(middle panel) or CA28-2GS-BF (50 ug/ml) (right panel). Flow cytometrywas performed after antibody staining using antibodies against CD59 andC3d. Results are shown in FIG. 13. In this figure, quadrant 1 (Q1) andquadrant 3 (Q3) represent Type III cells. Quadrant 2 (Q2) and quadrant 4(Q4) represent Types I and II cells. Q1 and Q2 represent cells with asignificant and abnormally high amount of C3 activation product (e.g.,C3d) deposition. Q3 and Q4 represent cells without significant C3ddeposition or somewhat elevated level (right portion of Q4) but less sothan Q2 cells. The percentages of cells in the different quadrants ispresented below each panel in FIG. 13 and in Table 13 below.

TABLE 13 No Inhibitor Eculizumab CA28-2GS-BF Popula- # % # % # % tionEvents Parent Events Parent Events Parent Q1 233 0.09 90,146 36.79 230.01 Q2 7,992 3.22 9,609 3.92 18 0.01 Q3 9,853 3.97 4,591 1.87 153,18761.54 Q4 230,241 92.72 140,689 57.42 95,713 38.45

As can be seen, in the absence of inhibitors the great majority of cellslie in Q4 (Type I or Type II with low levels of C3 activation productdeposition). Type III cells would have mainly been lysed, so theirpercentages (Q1 and Q3) are low. Q2 cells that accumulate C3 depositionproducts eventually lyse, so their number stays relatively low. In thepresence of eculizumab, Type III cells are protected from lysis at leastinitially, but accumulate C3 activation products (e.g., C3d) as shown bythe high percentage of Q1 cells as compared with the no inhibitor panel(36.79% vs 0.09%). The relative proportion of Q2+Q4 cells (Type I andII) is lower as a result of the increased survival of Type III cells.However, it is evident that significant deposition of C3 activationproducts (e.g., C3d) occurs on Type III cells, which may lead eventuallyto lysis or to clearance (in vivo). PNH RBCS treated with CA28-2GS-BF(right panel) exhibit essentially no deposition of C3d regardless ofwhether they are Type I, II, or III, in contrast to the results witheculizumab. The percentage of cells in Q1 and Q2 is negligible. There isa dramatic increase in the percentage of Type III cells (61.55%) ascompared with results with no inhibitor or with eculizumab, indicating(together with the lack of C3d deposition) enhanced protection fromlysis by CA28-2GS-BF.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. The scope of the presentinvention is not intended to be limited to the above Description, butrather is as set forth in the appended claims. It will be appreciatedthat the invention is in no way dependent upon particular resultsachieved in any specific example or with any specific embodiment.Articles such as “a”, “an” and “the” may mean one or more than oneunless indicated to the contrary or otherwise evident from the context.Claims or descriptions that include “or” between one or more members ofa group are considered satisfied if one, more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process unless indicated to the contrary or otherwiseevident from the context. The invention includes embodiments in whichexactly one member of the group is present in, employed in, or otherwiserelevant to a given product or process. For example, and withoutlimitation, it is understood that where claims or description indicatethat a residue at a particular position may be selected from aparticular group of amino acids or amino acid analogs, the inventionincludes individual embodiments in which the residue at that position isany of the listed amino acids or amino acid analogs. The invention alsoincludes embodiments in which more than one, or all of the group membersare present in, employed in, or otherwise relevant to a given product orprocess. Furthermore, it is to be understood that the inventionencompasses all variations, combinations, and permutations in which oneor more limitations, elements, clauses, descriptive terms, etc., fromone or more of the listed claims or from the description above isintroduced into another claim. For example, any claim that is dependenton another claim can be modified to include one or more elements,limitations, clauses, or descriptive terms, found in any other claimthat is dependent on the same base claim. Furthermore, where the claimsrecite a composition, it is to be understood that methods ofadministering the composition according to any of the methods disclosedherein, and methods of using the composition for any of the purposesdisclosed herein are included within the scope of the invention, andmethods of making the composition according to any of the methods ofmaking disclosed herein are included within the scope of the invention,unless otherwise indicated or unless it would be evident to one ofordinary skill in the art that a contradiction or inconsistency wouldarise. Methods of treating a subject can include a step of providing asubject in need of such treatment (e.g., a subject who has had, or is atincreased risk of having, a disease), a step of diagnosing a subject ashaving a disease and/or a step of selecting a subject for treatment witha cell-reactive compstatin analog. Where elements are presented aslists, it is to be understood that each subgroup of the elements is alsodisclosed, and any element(s) can be removed from the group. Forpurposes of conciseness only some of these embodiments have beenspecifically recited herein, but the invention includes all suchembodiments. It should also be understood that, in general, where theinvention, or aspects of the invention, is/are referred to as comprisingparticular elements, features, etc., certain embodiments of theinvention or aspects of the invention consist, or consist essentiallyof, such elements, features, etc. Discussion of various diseases,disorders, and conditions under various headings herein is forconvenience and is not intended to limit the invention.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of ordinary skill in the art, valuesthat are expressed as ranges can assume any specific value or subrangewithin the stated ranges in different embodiments of the invention, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise. Any particular embodiment, aspect,element, feature, etc., of the present invention may be explicitlyexcluded from the claims even if such exclusion is not set forthexplicitly herein. For example, any compstatin analog, functional group,linking portion, clearance-reducing moiety, disease, or indication canbe explicitly excluded.

We claim:
 1. A long-acting compstatin analog comprising aclearance-reducing moiety attached to two compstatin analog moieties,wherein each compstatin analog moiety comprises a cyclic peptideextended by a lysine residue or a sequence comprising a lysine residueat the N-terminus, C-terminus, or both, wherein the lysine residue isseparated from the cyclic portion of the peptide by a rigid or flexiblespacer comprising an oligo(ethylene glycol) moiety; the clearancereducing moiety comprises a linear polymer, wherein each end of thelinear polymer is linked to one of the compstatin analog moieties by wayof a carbamate; wherein (i) the spacer comprises —(CH2)_(m)— and—(O—CH2—CH2—)_(n) joined covalently, wherein m is between 1 and 10 and nis between 1 and 10; and/or (ii) the spacer comprises8-amino-3,6-dioxaoctanoic acid (AEEAc) or11-amino-3,6,9-trioxaundecanoic acid.
 2. The long-acting compstatinanalog of claim 1, wherein the cyclic peptide comprises an amino acidsequence as set forth in any of SEQ ID NOs: 3-36, 71, 72, 73, or
 74. 3.The long-acting compstatin analog of claim 1, wherein the spacercomprises —(CH2)_(m)— and —(O—CH2—CH2—)_(n) joined covalently, wherein mis between 1 and 10 and n is between 1 and
 10. 4. The long-actingcompstatin analog of claim 3, wherein m is 1 and n is
 2. 5. Thelong-acting compstatin analog of claim 1, wherein the spacer comprises8-amino-3,6-dioxaoctanoic acid (AEEAc) or11-amino-3,6,9-trioxaundecanoic acid.
 6. The long-acting compstatinanalog of claim 1, wherein the linear polymer is polyethylene glycol(PEG) or polyoxazoline (POZ).
 7. The long-acting compstatin analog ofclaim 6, wherein the linear polymer is PEG.
 8. The long-actingcompstatin analog of claim 7, wherein the PEG has an average molecularweight of about 40 kD.
 9. The long-acting compstatin analog of claim 1,having a terminal half-life of at least about 3 days when administeredto a primate.
 10. The long-acting compstatin analog of claim 9, having aterminal half-life of at least about 5 days when administered to aprimate.
 11. The long-acting compstatin analog of claim 1, having anactivity on a molar basis of at least 90% of that of a compstatin analogcomprising the same compstatin analog sequence as the compstatin analogmoiety but not linked to the clearance-reducing moiety.
 12. Thelong-acting compstatin analog of claim 2, wherein the cyclic peptidecomprises an amino acid sequence as set forth in SEQ ID NO: 28.